Promoter, promoter control elements, and combinations, and uses thereof

ABSTRACT

The present invention provides DNA molecules that constitute fragments of the genome of a plant, and polypeptides encoded thereby. The DNA molecules are useful for specifying a gene product in cells, either as a promoter or as a protein coding sequence or as an UTR or as a 3′ termination sequence, and are also useful in controlling the behavior of a gene in the chromosome, in controlling the expression of a gene or as tools for genetic mapping, recognizing or isolating identical or related DNA fragments, or identification of a particular individual organism, or for clustering of a group of organisms with a common trait. One of ordinary skill in the art, having this data, can obtain cloned DNA fragments, synthetic DNA fragments or polypeptides constituting desired sequences by recombinant methodology known in the art or described herein.

This application is a Continuation of application Ser. No. 10/886,468,filed Jul. 6, 2004, which is a Continuation of application Ser. No.10/653,278, filed on Sep. 3, 2003, which is a continuation-in-part ofthe following applications. The entire contents of which are herebyincorporated by reference:

Attorney Docket No. Filed Application Number 2750-1399P Feb. 1, 200109/775,870 2750-1549P Feb. 25, 2003 10/372,233 2750-1469P Aug. 9, 200109/924,702

Moreover,

Application Ser. No. 10/372,233 listed above is a continuation ofapplication Ser. No. 10/158,820 (Attorney No. 2750-1528P), filed on Jun.3, 2002, the entire contents of which are also hereby incorporated byreference. Application Ser. No. 10/158,820 (Attorney No. 2750-1528P) isa continuation of application Ser. No. 09/938,697 (Attorney No.2750-1468P), filed on Aug. 24, 2001, the entire contents of which arehereby incorporated by reference.

Application Ser. No. 09/938,697 (Attorney No. 2750-1468P) is acontinuation-in-part of the following non-provisional and provisionalapplications, to which the present application claims priority under 35USC §119(e) and §120, the entire contents of which are herebyincorporated by reference:

Attorney No Appln No Filed 2750-1154P 09/754,185 Jan. 5, 2001 2750-1398P09/774,340 Jan. 31, 2001 2750-1400P 09/776,014 Feb. 1, 2001 2750-1399P09/775,870 Feb. 1, 2001 2750-1439P 09/842,246 Apr. 26, 2001 2750-0832P60/200,034 Apr. 26, 2000 2750-1444P 09/845,209 May 1, 2001 2750-0889P60/205,233 May 17, 2000 2750-0841P 60/201,017 May 1, 2000 2750-1469P09/924,702 Aug. 9, 2001 2750-1114P 60/224,390 Aug. 9, 2000 2750-1181P60/228,208 Aug. 25, 2000 2750-1168P 60/228,052 Aug. 25, 2000 2750-1169P60/228,049 Aug. 25, 2000 2750-1170P 60/228,132 Aug. 25, 2000 2750-1171P60/228,152 Aug. 25, 2000 2750-1172P 60/228,135 Aug. 25, 2000 2750-1173P60/228,322 Aug. 25, 2000 2750-1174P 60/228,156 Aug. 25, 2000 2750-1175P60/228,323 Aug. 25, 2000 2750-1176P 60/228,133 Aug. 25, 2000 2750-1177P60/228,320 Aug. 25, 2000 2750-1178P 60/228,159 Aug. 25, 2000 2750-1167P60/228,047 Aug. 25, 2000 2750-1180P 60/228,202 Aug. 25, 2000 2750-1164P60/228,163 Aug. 25, 2000 2750-1182P 60/228,153 Aug. 25, 2000 2750-1183P60/228,179 Aug. 25, 2000 2750-1184P 60/228,180 Aug. 25, 2000 2750-1185P60/228,209 Aug. 25, 2000 2750-1187P 60/228,177 Aug. 25, 2000 2750-2132P60/227,791 Aug. 25, 2000 2750-1189P 60/228,207 Aug. 25, 2000 2750-1179P60/228,151 Aug. 25, 2000 2750-1144P 60/227,770 Aug. 25, 2000 2750-1133P60/228,025 Aug. 25, 2000 2750-1134P 60/227,781 Aug. 25, 2000 2750-1135P60/227,783 Aug. 25, 2000 2750-1136P 60/227,731 Aug. 25, 2000 2750-1137P60/227,732 Aug. 25, 2000 2750-1138P 60/227,729 Aug. 25, 2000 2750-1139P60/228,167 Aug. 25, 2000 2750-1140P 60/227,734 Aug. 25, 2000 2750-1141P60/227,792 Aug. 25, 2000 2750-1166P 60/228,098 Aug. 25, 2000 2750-1143P60/227,730 Aug. 25, 2000 2750-1190P 60/228,048 Aug. 25, 2000 2750-1145P60/227,728 Aug. 25, 2000 2750-1146P 60/227,773 Aug. 25, 2000 2750-1147P60/228,033 Aug. 25, 2000 2750-1148P 60/228,024 Aug. 25, 2000 2750-1149P60/227,769 Aug. 25, 2000 2750-1150P 60/227,780 Aug. 25, 2000 2750-1151P60/227,725 Aug. 25, 2000 2750-1152P 60/227,774 Aug. 25, 2000 2750-1188P60/227,976 Aug. 25, 2000 2750-1165P 60/228,046 Aug. 25, 2000 2750-1142P60/227,733 Aug. 25, 2000 2750-1218P 60/227,929 Aug. 25, 2000 2750-1191P60/228,096 Aug. 25, 2000 2750-1220P 60/227,931 Aug. 25, 2000 2750-1186P60/228,178 Aug. 25, 2000 2750-1222P 60/228,061 Aug. 25, 2000 2750-1223P60/228,150 Aug. 25, 2000 2750-2133P 60/228,041 Aug. 25, 2000 2750-2116P60/227,793 Aug. 25, 2000 2750-2117P 60/228,031 Aug. 25, 2000 2750-1217P60/228,217 Aug. 25, 2000 2750-2119P 60/228,027 Aug. 25, 2000 2750-1219P60/228,043 Aug. 25, 2000 2750-2121P 60/228,026 Aug. 25, 2000 2750-2122P60/228,038 Aug. 25, 2000 2750-2123P 60/228,036 Aug. 25, 2000 2750-2124P60/227,790 Aug. 25, 2000 2750-2125P 60/228,039 Aug. 25, 2000 2750-2126P60/228,030 Aug. 25, 2000 2750-2127P 60/228,032 Aug. 25, 2000 2750-2128P60/228,149 Aug. 25, 2000 2750-2129P 60/228,040 Aug. 25, 2000 2750-2130P60/227,777 Aug. 25, 2000 2750-2131P 60/228,037 Aug. 25, 2000 2750-2118P60/228,028 Aug. 25, 2000 2750-1201P 60/228,055 Aug. 25, 2000 2750-1192P60/227,932 Aug. 25, 2000 2750-1193P 60/227,936 Aug. 25, 2000 2750-1194P60/228,044 Aug. 25, 2000 2750-1195P 60/228,216 Aug. 25, 2000 2750-1196P60/228,065 Aug. 25, 2000 2750-1197P 60/227,975 Aug. 25, 2000 2750-1198P60/228,181 Aug. 25, 2000 2750-1221P 60/228,187 Aug. 25, 2000 2750-1200P60/228,064 Aug. 25, 2000 2750-1216P 60/227,954 Aug. 25, 2000 2750-1202P60/228,074 Aug. 25, 2000 2750-1203P 60/227,939 Aug. 25, 2000 2750-1212P60/228,165 Aug. 25, 2000 2750-1213P 60/228,221 Aug. 25, 2000 2750-1199P60/228,063 Aug. 25, 2000 2750-1214P 60/228,240 Aug. 25, 2000 2750-1204P60/227,955 Aug. 25, 2000 2750-1211P 60/228,161 Aug. 25, 2000 2750-1210P60/228,164 Aug. 25, 2000 2750-1209P 60/228,054 Aug. 25, 2000 2750-1208P60/228,189 Aug. 25, 2000 2750-1207P 60/227,982 Aug. 25, 2000 2750-1206P60/227,978 Aug. 25, 2000 2750-1205P 60/228,053 Aug. 25, 2000 2750-1215P60/227,979 Aug. 25, 2000

Application Ser. No. 09/924,702 listed above claims priority under 35USC §119(e) of the following application. The entire contents of whichare hereby incorporated by reference.

Country Filed Attorney No. Application No. United States Aug. 9, 20002750-1114P 60/224,390

The entire contents of the applications listed in the table above areexpressly incorporated herein by reference.

This application contains a CDR, the entire contents of which are herebyincorporated by reference. The CDR contains the following files:

Create Date: File Size: File Name: 09/26/2002 09:19p 2,366,9532750-1399P Table A-1.txt 09/26/2002 09:19p 1,938,556 2750-1399P TableA-2.txt 09/26/2002 09:19p 74,754,025 2750-1399P Table B.txt 09/26/200209:19p 7,670,452 2750-1399P Table C.txt 09/26/2002 06:08p 65,168,2042750-1469P.txt 09/26/2002 05:46p 4,052,876 cdna_clusters.txt 09/26/200205:46p 399,505 cDNA_GI_pos.txt 09/26/2002 05:46p 35,153 ClusterFunctions and Utilities (01).txt 09/26/2002 05:46p 40,447 ClusterFunctions and Utilities (02).txt 09/26/2002 05:46p 4,473 ClusterFunctions and Utilities (03).txt 09/26/2002 05:46p 7,820 ClusterFunctions and Utilities (04).txt 09/26/2002 05:46p 24,047 ClusterFunctions and Utilities (05).txt 09/26/2002 05:46p 18,490 ClusterFunctions and Utilities (06).txt 09/26/2002 05:46p 36,273 Clusterfunctions and utilities (07).txt 09/26/2002 05:46p 33,962 ClusterFunctions and Utilities (08).txt 09/26/2002 05:46p 23,000 Clusterfunctions and utilities (09).txt 09/26/2002 05:46p 2,691 Clusterfunctions and utilities (10).txt 09/26/2002 05:46p 2,290 Clusterfunctions and utilities (11).txt 09/26/2002 05:46p 23,740 ClusterFuntions and Utilities (12).txt 09/26/2002 05:46p 6,642,415cluster_info_50.dat 09/26/2002 05:46p 2,946,892 cluster_info_60.dat09/26/2002 05:46p 913,656 cluster_info_70.dat 09/26/2002 05:46p 432,906cluster_info_75.dat 09/26/2002 05:46p 392,675knock_in.710-0024-55300-US-U-00007_01.txt 09/26/2002 05:46p 831,736knock_out.710-0024-55300-US-U-00007.01 09/26/2002 05:46p 5,328,883MATRIX.001 09/26/2002 05:46p 5,335,846 MATRIX.002 09/26/2002 05:46p5,330,456 MATRIX.003 09/26/2002 05:46p 5,317,696 MATRIX.004 09/26/200205:46p 605,072 MATRIX.005 09/26/2002 05:46p 16,635,460ma_clusters.710-0024-55300-US-U-00007.01 09/26/2002 05:46p 55,307ma_diff Aluminum.txt 09/26/2002 05:46p 27,557 ma_diff Axel.txt09/26/2002 05:46p 41,505 ma_diff Cadium .txt 09/26/2002 05:46p 53,938ma_diff Cauliflower .txt 09/26/2002 05:46p 98,775 ma_diffChloroplast.txt 09/26/2002 05:46p 141,971 ma_diff Circadian 1-01.txt09/26/2002 05:46p 160,542 ma_diff Circadian 1-02.txt 09/26/2002 05:46p127,498 ma_diff Circadian 1-03.txt 09/26/2002 05:46p 166,158 ma_diffCircadian 1-04.txt 09/26/2002 05:46p 56,536 ma_diff Circadian 1-05.txt09/26/2002 05:46p 121,178 ma_diff Circadian 1-06.txt 09/26/2002 05:46p133,389 ma_diff Circadian 1-07.txt 09/26/2002 05:46p 259,096 ma_diffCircadian 1-08.txt 09/26/2002 05:46p 228,222 ma_diff Circadian 1-09.txt09/26/2002 05:46p 54,526 ma_diff Circadian 1-10.txt 09/26/2002 05:46p134,759 ma_diff CO2 1-1.txt 09/26/2002 05:46p 241,865 ma_diff CO21-2.txt 09/26/2002 05:46p 63,264 ma_diff CO2 1-3.txt 09/26/2002 05:46p59,530 ma_diff CO2 1-4.txt 09/26/2002 05:46p 372,633 ma_diff CO2 1-5.txt09/26/2002 05:46p 9,220 ma_diff Disease .txt 09/26/2002 05:46p 25,114ma_diff H2O2 .txt 09/26/2002 05:46p 4,073 ma_diff Iol .txt 09/26/200205:46p 283,026 ma_diff Iron 1-1.txt 09/26/2002 05:46p 90,890 ma_diffIron 1-2.txt 09/26/2002 05:46p 51,342 ma_diff Mitochondria-ElectronTransp.txt 09/26/2002 05:46p 107,920 ma_diff NAA (Auxin) 1-1.txt09/26/2002 05:46p 50,267 ma_diff NAA (Auxin) 1-2.txt 09/26/2002 05:46p67,291 ma_diff Nitrogen.txt 09/26/2002 05:46p 6,441 ma_diff Phototropism1-1.txt 09/26/2002 05:46p 22,229 ma_diff Phototropism 1-2.txt 09/26/200205:46p 28,270 ma_diff Phototropism 1-3.txt 09/26/2002 05:46p 45,620ma_diff Shade.txt 09/26/2002 05:46p 73,438 ma_diff Sqn.txt 09/26/200205:46p 3,828 ma_diff Sulfur.txt 09/26/2002 05:46p 67,949 ma_diffWounding.txt 09/26/2002 05:46p 30,836 ma_diff Zinc.txt 09/26/2002 05:46p4,318,956 ma_diff.710-0024-55300-US-U-00007.01 09/26/2002 05:46p1,071,693 RESULT.001 09/26/2002 05:46p 1,072,021 RESULT.002 09/26/200205:46p 1,072,022 RESULT.003 09/26/2002 05:46p 1,071,371 RESULT.00409/26/2002 05:46p 1,071,155 RESULT.005 09/26/2002 05:46p 459,530RESULT.006 09/26/2002 05:46p 1,476 Single gene functions and utilities(1).txt 09/26/2002 05:46p 2,223 Single gene functions and utilities(2).txt 09/26/2002 05:46p 905 Single gene functions and utilities(3).txt 09/26/2002 05:46p 1,517 Single gene functions and utilities(4).txt 09/26/2002 05:46p 4,626 Single gene functions and utilities(5).txt 09/26/2002 05:46p 4,887 Single gene functions and utilities(6).txt 09/26/2002 05:46p 7,456 Single gene functions and utilities(7).txt 09/26/2002 05:46p 9,339 Single gene functions and utilities(8).txt 09/26/2002 05:46p 228,792 stanford_old_new_cdna_map.txt

FIELD OF THE INVENTION

The present invention relates to over 100,000 isolated polynucleotidesfrom plants that include a complete coding sequence, or a fragmentthereof, that is expressed. In addition, the present invention relatesto the polypeptide or protein corresponding to the coding sequence ofthese polynucleotides. The present invention also relates to isolatedpolynucleotides that represent regulatory regions of genes. The presentinvention also relates to isolated polynucleotides that representuntranslated regions of genes. The present invention further relates tothe use of these isolated polynucleotides and polypeptides and proteins.

BACKGROUND OF THE INVENTION

There are more than 300,000 species of plants. They show a widediversity of forms, ranging from delicate liverworts, adapted for lifein a damp habitat, to cacti, capable of surviving in the desert. Theplant kingdom includes herbaceous plants, such as corn, whose life cycleis measured in months, to the giant redwood tree, which can live forthousands of years. This diversity reflects the adaptations of plants tosurvive in a wide range of habitats. This is seen most clearly in theflowering plants (phylum Angiospermophyta), which are the most numerous,with over 250,000 species. They are also the most widespread, beingfound from the tropics to the arctic.

The process of plant breeding involving man's intervention in naturalbreeding and selection is some 20,000 years old. It has producedremarkable advances in adapting existing species to serve new purposes.The world's economics was largely based on the successes of agriculturefor most of these 20,000 years.

Plant breeding involves choosing parents, making crosses to allowrecombination of gene (alleles) and searching for and selecting improvedforms. Success depends on the genes/alleles available, the combinationsrequired and the ability to create and find the correct combinationsnecessary to give the desired properties to the plant. Moleculargenetics technologies are now capable of providing new genes, newalleles and the means of creating and selecting plants with the new,desired characteristics.

When the molecular and genetic basis for different plant characteristicsare understood, a wide variety of polynucleotides, both endogenouspolynucleotides and created variants, polypeptides, cells, and wholeorganisms, can be exploited to engineer old and new plant traits in avast range of organisms including plants. These traits can range fromthe observable morphological characteristics, through adaptation tospecific environments to biochemical composition and to molecules thatthe plants (organisms) exude. Such engineering can involve tailoringexisting traits, such as increasing the production of taxol in yewtrees, to combining traits from two different plants into a singleorganism, such as inserting the drought tolerance of a cactus into acorn plant. Molecular and genetic knowledge also allows the creation ofnew traits. For example, the production of chemicals and pharmaceuticalsthat are not native to particular species or the plant kingdom as awhole.

The application reports the inventions Applicants have discovered tobuild a foundation of scientific understanding of plant genomes toachieve these aims. These inventions include polynucleotide andpolypeptide sequences, and data relating to where and when the genes aredifferentially expressed and phenotypic observations resulting fromeither aberrant gene activation or disruption. How these data aretransformed into a scientific understanding of plant biology and thecontrol of traits from a genetic perspective also is explained by theinstant application. Applications of these discoveries to create newprototypes and products in the field of chemical, pharmaceutical, food,feed, and fiber production are described herein as well.

The achievements described in this application were possible because ofthe results from a cluster of technologies, a genomic engine, depictedbelow in Schematic 1, that allows information on each gene to beintegrated to provide a more comprehensive understanding of genestructure and function and the deployment of genes and gene componentsto make new products.

I. The Discoveries of the Instant Application

Applicants have isolated and identified over one hundred thousand genes,gene components and their products and thousands of promoters. Specificgenes were isolated and/or characterized from arabidopsis, soybean,maize, wheat and rice. These species were selected because of theireconomic value and scientific importance and were deliberately chosen toinclude representatives of the evolutionary divergent dicotyledonous andmonocotyledonous groups of the plant kingdom. The number of genescharacterized in this application represents a large proportion of allthe genes in these plant species.

The techniques used initially to isolate and characterize most of thegenes, namely sequencing of full-length cDNAs, were deliberately chosento provide information on complete coding sequences and on the completesequences of their protein products.

Gene components and products the Applicants have identified includeexons, introns, promoters, coding sequences, antisense sequences,terminators and other regulatory sequences. The exons are characterizedby the proteins they encode and arabidopsis promoters are characterizedby their position in the genomic DNA relative to where mRNA synthesisbegins and in what cells and to what extent they promote mRNA synthesis.Further exploitation of molecular genetics technologies has helped theApplicants to understand the functions and characteristics of each geneand their role in a plant. Three powerful molecular genetics approacheswere used to this end:

-   -   (a) Analyses of the phenotypic changes when the particular gene        sequence is interrupted or activated differentially;        (arabidopsis)    -   (b) Analyses of in what plant organs, to what extent, and in        response to what environmental signals mRNA is synthesized from        the gene; (arabidopsis and maize) and    -   (c) Analysis of the gene sequence and its relatives. (all        species)

These were conducted using the genomics engine depicted in FIG. 1 thatallows information on each gene to be integrated to provide a morecomprehensive understanding of gene structure and function and linkageto potential products.

The species arabidopsis was used extensively in these studies forseveral reasons: (1) the complete genomic sequence, though poorlyannotated in terms of gene recognition, was being produced and publishedby others and (2) genetic experiments to determine the role of the genesin planta are much quicker to complete.

The phenotypic tables, MA tables, and reference tables and sequencetables indicate the results of these analyses and thus the specificfunctions and characteristics that are ascribed to the genes and genecomponents and products.

II. Integration of Discoveries to Provide Scientific Understanding

From the discoveries made, Applicants have deduced the biochemicalactivities, pathways, cellular roles, and developmental andphysiological processes that can be modulated using these components.These are discussed and summarized in sections based on the genefunctions characteristics from the analyses and role in determiningphenotypes. These sections illustrate and emphasize that each gene, genecomponent or product influences biochemical activities, cells ororganisms in complex ways, from which there can be many phenotypicconsequences.

An illustration of how the discoveries on gene structure, function,expression and phenotypic observation can be integrated together tounderstand complex phenotypes is provided in FIG. 2. This sort ofunderstanding enables conclusions to be made as to how the genes, genecomponents and product are useful for changing the properties of plantsand other organisms. This example also illustrates how single genechanges in, for example, a metabolic pathway can cause gross phenotypicchanges.

Furthermore, the development and properties of one part of plant can beinterconnected with other parts. The dependence of shoot and leafdevelopment on root cells is a classic example. Here, shoot growth anddevelopment require nutrients supplied from roots, so the proteincomplement of root cells can affect plant development, including flowersand seed production. Similarly, root development is dependent on theproducts of photosynthesis from leaves. Therefore, proteins in leavescan influence root developmental physiology and biochemistry.

Thus, the following sections describe both the functions andcharacteristics of the genes, gene components and products and also themultiplicity of biochemical activities, cellular functions, and thedevelopmental and physiological processes influenced by them. Thesections also describe examples of commercial products that can berealized from the inventions.

A. Analyses to Reveal Function and In Vivo Roles of Single Genes in OnePlant Species

The genomics engine has focused on individual genes to reveal themultiple functions or characteristics that are associated to each gene,gene components and products of the instant invention in the livingplant. For example, the biochemical activity of a protein is deducedbased on its similarity to a protein of known function. In this case,the protein may be ascribed with, for example, an oxidase activity.Where and when this same protein is active can be uncovered fromdifferential expression experiments, which show that the mRNA encodingthe protein is differentially expressed in response to drought and inseeds but not roots. The gene disruption experiments reveal that absenceof the same protein causes embryo lethality.

Thus, this protein is characterized as a seed protein anddrought-responsive oxidase that is critical for embryo viability.

B. Analyses to Reveal Function and Roles of Single Genes in DifferentSpecies

The genomics engine has also been used to extrapolate knowledge from onespecies to many plant species. For example, proteins from differentspecies, capable of performing identical or similar functions, preservemany features of amino acid sequence and structure during evolution.Complete protein sequences have been compared and contrasted within andbetween species to determine the functionally vital domains andsignatures characteristic of each of the proteins that is the subject ofthis application. Thus, functions and characteristics of arabidopsisproteins have been extrapolated to proteins containing similar domainsand signatures of corn, soybean, rice and wheat and by implication toall other (plant) species.

Schematic 3 provides an example. Two proteins with related structures,one from corn, a monocot, and one from arabidopsis, a dicot, have beenconcluded to be orthologs. The known characteristics of the arabidopsisprotein (seed protein, drought responsive oxidase) can then beattributed to the corn protein.

C. Analyses Over Multiple Experiments to Reveal Gene Networks and LinksAcross Species

The genomics engine can identify networks or pathways of genes concernedwith the same process and hence linked to the same phenotype(s). Genesspecifying functions of the same pathway or developmental environmentalresponses are frequently co-regulated i.e. they are regulated bymechanisms that result in coincident increases or decreases for all genemembers in the group. The Applicants have divided the genes ofarabidopsis and maize into such co-regulated groups on the basis oftheir expression patterns and the function of each group has beendeduced. This process has provided considerable insight into thefunction and role of thousands of the plant genes in diverse speciesincluded in this application.

D. Applications of Applicant's Discoveries

It will be appreciated while reading the sections that the differentexperimental molecular genetic approaches focused on different aspectsof the pathway from gene and gene product through to the properties oftissues, organs and whole organisms growing in specific environments.For each endogenous gene, these pathways are delineated within theexisting biology of the species. However, Applicants' inventions allowgene components or products to be mixed and matched to create new genesand placed in other cellular contexts and species, to exhibit newcombinations of functions and characteristics not found in nature, or toenhance and modify existing ones. For instance, gene components can beused to achieve expression of a specific protein in a new cell type tointroduce new biochemical activities, cellular attributes ordevelopmental and physiological processes. Such cell-specific targetingcan be achieved by combining polynucleotides encoding proteins with anyone of a large array of promoters to facilitate synthesis of proteins ina selective set of plant cells. This emphasizes that each gene,component and protein can be used to cause multiple and differentphenotypic effects depending on the biological context. The utilitiesare therefore not limited to the existing in vivo roles of the genes,gene components, and gene products.

While the genes, gene components and products disclosed herein can actalone, combinations are useful to modify or modulate different traits.Useful combinations include different polynucleotides and/or genecomponents or products that have (1) an effect in the same or similardevelopmental or biochemical pathways; (2) similar biologicalactivities; (3) similar transcription profiles; or (4) similarphysiological consequences.

Of particular interest are the transcription factors and key factors inregulatory transduction pathways, which are able to control entirepathways, segments of pathways or large groups of functionally relatedgenes. Therefore, manipulation of such proteins, alone or in combinationis especially useful for altering phenotypes or biochemical activitiesin plants. Because interactions exist between hormone, nutrition, anddevelopmental pathways, combinations of genes and/or gene products fromthese pathways also are useful to produce more complex changes. Inaddition to using polynucleotides having similar transcription profilesand/or biological activities, useful combinations includepolynucleotides that may exhibit different transcription profiles butwhich participate in common or overlapping pathways. Also,polynucleotides encoding selected enzymes can be combined in novel waysin a plant to create new metabolic pathways and hence new metabolicproducts.

The utilities of the various genes, gene components and products of theApplication are described below in the sections entitled as follows:

I. Organ Affecting Genes, Gene Components, Products (IncludingDifferentiation Function)

I.A. Root Genes, Gene Components And Products

-   -   I.A.1. Root Genes, Gene Components And Products    -   I.A.2. Root Hair Genes, Gene Components And Products

I.B. Leaf Genes, Gene Components And Products

-   -   I.B.1. Leaf Genes, Gene Components And Products    -   I.B.2. Trichome Genes And Gene Components    -   I.B.3. Chloroplast Genes And Gene Components

I.C. Reproduction Genes, Gene Components And Products

-   -   I.C.1. Reproduction Genes, Gene Components And Products    -   I.C.2. Ovule Genes, Gene Components And Products    -   I.C.3. Seed And Fruit Development Genes, Gene Components And        Products

I.D. Development Genes, Gene Components And Products

-   -   I.D.1. Imbibition and Germination Responsive Genes, Gene        Components And Products    -   I.D.2. Early Seedling Phase Genes, Gene Components And Products    -   I.D.3. Size and Stature Genes, Gene Components And Products    -   I.D.4. Shoot-Apical Meristem Genes, Gene Components And Products    -   I.D.5. Vegetative-Phase Specific Responsive Genes, Gene        Components And Products

II. Hormones Responsive Genes, Gene Components And Products

II.A. Abscissic Acid Responsive Genes, Gene Components And Products

II.B. Auxin Responsive Genes, Gene Components And Products

II.C. Brassinosteroid Responsive Genes, Gene Components And Products

II.D. Cytokinin Responsive Genes, Gene Components And Products

II.E. Gibberellic Acid Responsive Genes, Gene Components And Products

III. Metabolism Affecting Genes, Gene Components And Products

III.A. Nitrogen Responsive Genes, Gene Components And Products

III.B. Circadian Rhythm Responsive Genes, Gene Components And Products

III.C. Blue Light (Phototropism) Responsive Genes, Gene Components AndProducts

III.D. Co2 Responsive Genes, Gene Components And Products

III.E. Mitochondria Electron Transport Genes, Gene Components AndProducts

III.F. Protein Degradation Genes, Gene Components And Products

III.G. Carotenogenesis Responsive Genes, Gene Components And Products

IV. Viability Genes, Gene Components And Products

IV.A. Viability Genes, Gene Components And Products

IV.B. Histone Deacetylase (Axel) Responsive Genes, Gene Components AndProducts

V. Stress Responsive Genes, Gene Components And Products

V.A. Cold Responsive Genes, Gene Components And Products

V.B. Heat Responsive Genes, Gene Components And Products

V.C. Drought Responsive Genes, Gene Components And Products

V.D. Wounding Responsive Genes, Gene Components And Products

V.E. Methyl Jasmonate Responsive Genes, Gene Components And Products

V.F. Reactive Oxygen Responsive Genes, Gene Components And H2O2 Products

V.G. Salicylic Acid Responsive Genes, Gene Components And Products

V.H. Nitric Oxide Responsive Genes, Gene Components And Products

V.I. Osmotic Stress Responsive Genes, Gene Components And Products

V.J. Aluminum Responsive Genes, Gene Components And Products

V.K. Cadmium Responsive Genes, Gene Components And Products

V.L. Disease Responsive Genes, Gene Components And Products

V.M. Defense Responsive Genes, Gene Components And Products

V.N. Iron Responsive Genes, Gene Components And Products

V.O. Shade Responsive Genes, Gene Components And Products

V.P. Sulfur Responsive Genes, Gene Components And Products

V.Q. Zinc Responsive Genes, Gene Components And Products

VI. Enhanced Foods VII. Pharmaceutical Products VIII. Precursors OfIndustrial Scale Compounds IX. Promoters As Sentinels SUMMARY OF THEINVENTION

The present invention comprises polynucleotides, such as complete cDNAsequences and/or sequences of genomic DNA encompassing complete genes,fragments of genes, and/or regulatory elements of genes and/or regionswith other functions and/or intergenic regions, hereinafter collectivelyreferred to as Sequence-Determined DNA Fragments (SDFs) or sometimescollectively referred to as “genes or gene components”, or sometimes as“genes, gene components or products”, from different plant species,particularly corn, wheat, soybean, rice and Arabidopsis thaliana, andother plants and or mutants, variants, fragments or fusions of said SDFsand polypeptides or proteins derived therefrom. In some instances, theSDFs span the entirety of a protein-coding segment. In some instances,the entirety of an mRNA is represented. Other objects of the inventionthat are also represented by SDFs of the invention are controlsequences, such as, but not limited to, promoters. Complements of anysequence of the invention are also considered part of the invention.

Other objects of the invention are polynucleotides comprising exonsequences, polynucleotides comprising intron sequences, polynucleotidescomprising introns together with exons, intron/exon junction sequences,5′ untranslated sequences, and 3′ untranslated sequences of the SDFs ofthe present invention. Polynucleotides representing the joinder of anyexons described herein, in any arrangement, for example, to produce asequence encoding any desirable amino acid sequence are within the scopeof the invention.

The present invention also resides in probes useful for isolating andidentifying nucleic acids that hybridize to an SDF of the invention. Theprobes can be of any length, but more typically are 12-2000 nucleotidesin length; more typically, 15 to 200 nucleotides long; even moretypically, 18 to 100 nucleotides long.

Yet another object of the invention is a method of isolating and/oridentifying nucleic acids using the following steps:

(a) contacting a probe of the instant invention with a polynucleotidesample under conditions that permit hybridization and formation of apolynucleotide duplex; and

(b) detecting and/or isolating the duplex of step (a).

The conditions for hybridization can be from low to moderate to highstringency conditions. The sample can include a polynucleotide having asequence unique in a plant genome. Probes and methods of the inventionare useful, for example, without limitation, for mapping of genetictraits and/or for positional cloning of a desired fragment of genomicDNA.

Probes and methods of the invention can also be used for detectingalternatively spliced messages within a species. Probes and methods ofthe invention can further be used to detect or isolate related genes inother plant species using genomic DNA (gDNA) and/or cDNA libraries. Insome instances, especially when longer probes and low to moderatestringency hybridization conditions are used, the probe will hybridizeto a plurality of cDNA and/or gDNA sequences of a plant. This approachis useful for isolating representatives of gene families which areidentifiable by possession of a common functional domain in the geneproduct or which have common cis-acting regulatory sequences. Thisapproach is also useful for identifying orthologous genes from otherorganisms.

The present invention also resides in constructs for modulating theexpression of the genes comprised of all or a fragment of an SDF. Theconstructs comprise all or a fragment of the expressed SDF, or of acomplementary sequence. Examples of constructs include ribozymescomprising RNA encoded by an SDF or by a sequence complementary thereto,antisense constructs, constructs comprising coding regions or partsthereof, constructs comprising promoters, introns, untranslated regions,scaffold attachment regions, methylating regions, enhancing or reducingregions, DNA and chromatin conformation modifying sequences, etc. Suchconstructs can be constructed using viral, plasmid, bacterial artificialchromosomes (BACs), plasmid artificial chromosomes (PACs), autonomousplant plasmids, plant artificial chromosomes or other types of vectorsand exist in the plant as autonomous replicating sequences or as DNAintegrated into the genome. When inserted into a host cell the constructis, preferably, functionally integrated with, or operatively linked to,a heterologous polynucleotide. For instance, a coding region from an SDFmight be operably linked to a promoter that is functional in a plant.

The present invention also resides in host cells, including bacterial oryeast cells or plant cells, and plants that harbor constructs such asdescribed above. Another aspect of the invention relates to methods formodulating expression of specific genes in plants by expression of thecoding sequence of the constructs, by regulation of expression of one ormore endogenous genes in a plant or by suppression of expression of thepolynucleotides of the invention in a plant. Methods of modulation ofgene expression include without limitation (1) inserting into a hostcell additional copies of a polynucleotide comprising a coding sequence;(2) modulating an endogenous promoter in a host cell; (3) insertingantisense or ribozyme constructs into a host cell and (4) inserting intoa host cell a polynucleotide comprising a sequence encoding a variant,fragment, or fusion of the native polypeptides of the instant invention.

DETAILED DESCRIPTION OF THE INVENTION I. Description of the Tables

As noted above, the Applicants have obtained and analyzed an extensiveamount of information on a large number of genes by use of the CeresGenomic Engine to determine. This information can be categorized intothree basic types:

A. Sequence Information for the Inventions

B. Transcriptional Information for the Inventions

C. Phenotypic Information for the Inventions

I.A. Sequence Information

To harness the potential of the plant genome, Applicants began byelucidating a large number gene sequences, including the sequences ofgene components and products, and analyzing the data. The list ofsequences and associated data are presented in the Reference andSequence Tables of the present application (sometimes referred to as the“REF” and “SEQ” Tables). The Reference and Sequence tables include:

-   -   cDNA sequence;    -   coding sequence;    -   5′ & 3′ UTR;    -   transcription start sites;    -   exon and intron boundaries in genomic sequence; and    -   protein sequence.

The Reference and Sequence Tables also include computer-based,comparative analyses between the protein sequences of the invention andsequences with known function. Proteins with similar sequences typicallyexhibit similar biochemical activities. The Reference table notes:

-   -   sequences of known function that are similar to the Applicants'        proteins; and    -   biochemical activity that is associated with Applicants'        proteins.

Also, by analyzing the protein sequences, Applicants were able to groupthe protein sequences into groups, wherein all the sequences in thegroup contain a signature sequence. The groups are presented in theProtein Group Table. The signature sequences are reported in the ProteinGroup Table. More detailed analyses of the signature sequences are shownin the Protein Group Matrix Table.

To identify gene components and products, Applicants took a cDNA/codingsequence approach. That is, Applicants initiated their studies either byisolating cDNAs and determining their sequences experimentally, or byidentifying the coding sequence from genomic sequence with the aid ofpredictive algorithms. The cDNA sequences and coding sequences also arereferred to as “Maximum Length Sequences” in the Reference tables. ThecDNA and coding sequences were given this designation to indicate thesewere the maximum length of coding sequences identified by Applicants.

Due to this cDNA/coding sequence focus of the present application, theReference and Sequence Tables were organized around cDNA and codingsequences. Each of these Maximum Length Sequences was assigned a uniqueidentifier: Ceres Sequence ID NO, which is reported in the Tables.

All data that relate to these Maximum Length Sequences are groupedtogether, including 5′ & 3′ UTRs; transcription start sites; exon andintron boundaries in genomic sequence; protein sequence, etc.

Below, a more detailed explanation of the organization of the Referenceand Sequence Tables and how the data in the tables were generated isprovided.

a. cDNA

Applicants have ascertained the sequences of mRNAs from differentorganisms by reverse transcription of mRNA to DNA, which was cloned andthen sequenced. These complementary DNA or cDNA sequences also arereferred to as Maximum Length Sequences in the Reference Tables, whichcontain details on each of the sequences in the Sequence Tables.

Each sequence was assigned a Pat. Appln. Sequence ID NO: and an internalCeres Sequence ID NO: as reported in the Reference Table, the sectionlabeled “(Ac) cDNA Sequence.” An example is shown below:

Max Len. Seq.:

(Ac) cDNA Sequence

-   -   Pat. Appln. Sequence ID NO: 174538    -   Ceres Sequence ID NO: 5673127

Both numbers are included in the Sequence Table to aid in tracking ofinformation, as shown below:

<210> 174538 (Pat. Appln. Sequence ID NO:) <211> 1846 <212>DNA (genomic) <213> Arabidopsis thaliana <220> <221> misc feature <222>(1)..(1846) <223> Ceres Seq. ID no. 5673127 <220> <221> misc_feature<222> ( )..( ) <223> n is a, c, t, g, unknown, or other <400> 174538acaagaacaa caaaacagag gaagaagaag aagaagatga agcttctggc tctgtttcca 60tttctagcga tcgtgatcca actcagctgt... etc.

The Sequence and Reference Tables are divided into sections by organism:Arabidopsis thaliana, Brassica napus, Glycine max, Zea mays, Triticumaestivum; and Oryza sativa.

b. Coding Sequence

The coding sequence portion of the cDNA was identified by usingcomputer-based algorithms and comparative biology. The sequence of eachcoding sequence of the cDNA is reported in the “PolyP Sequence” sectionof the Reference Tables, which are also divided into sections byorganism. An example shown below for the peptides that relate to thecDNA sequence above

PolyP Sequence

-   -   Pat. Appln. Sequence ID NO 174539    -   Ceres Sequence ID NO 5673128    -   Loc. Sequence ID NO 174538: @ 1 nt.    -   Loc. Sig. P. Sequence ID NO 174539: @ 37 aa.        The polypeptide sequence can be found in the Sequence Tables by        either the Pat. Appln. Sequence ID NO or by the Ceres Sequence        ID NO: as shown below:

<210> 174539 (Pat. Appln. Sequence ID NO) <211> 443 <212> PRT <213>Arabidopsis thaliana <220> <221> peptide <222> (1)..(443) <223>Ceres Seq. ID no. 5673128 <220> <221> misc feature <222> ( )..( ) <223>xaa is any aa, unknown or other <400> 174539Thr Arg Thr Thr Lys Gln Arg Lys Lys Lys Lys Lys1         5           10          15 Met Lys Leu Leu Ala Leu Phe Pro Phe                                25 Leu Ala Ile... etc.

The PolyP section also indicates where the coding region begins in theMaximum Length Sequence. More than one coding region may be indicatedfor a single polypeptide due to multiple potential translation startcodons. Coding sequences were identified also by analyzing genomicsequence by predictive algorithms, without the actual cloning of a cDNAmolecule from a mRNA. By default, the cDNA sequence was considered thesame as the coding sequence, when Maximum Length Sequence was splicedtogether from a genomic annotation.

c. 5′ and 3′ UTR

The 5′ UTR can be identified as any sequence 5′ of the initiating codonof the coding sequence in the cDNA sequence. Similarly, the 3′ UTR isany sequence 3′ of the terminating codon of the coding sequence.

d. Transcription Start Sites

Applicants cloned a number of cDNAs that encompassed the same codingsequence but comprised 5′ UTRs of different lengths. These differentlengths revealed the multiple transcription start sites of the gene thatcorresponded to the cDNA. These multiple transcription start sites arereported in the “Sequence # w. TSS” section” of the Reference Tables.

e. Exons & Introns

Alignment of the cDNA sequences and coding portions to genomic sequencepermitted Applicants to pinpoint the exon/intron boundaries. Theseboundaries are identified in the Reference Table under the “Pub gDNA”section. That section reports the gi number of the public BAC sequencethat contains the introns and exons of interest. An example is shownbelow:

Max Len. Seq.:

Pub gDNA:

-   -   gi No: 1000000005    -   Gen. seq. in cDNA:        -   115777 . . . 115448 by Method #1        -   115105 . . . 114911 by Method #1        -   114822 . . . 114700 by Method #1        -   114588 . . . 114386 by Method #1        -   114295 . . . 113851 by Method #1        -   115777 . . . 115448 by Method #2        -   115105 . . . 114911 by Method #2        -   114822 . . . 114700 by Method #2        -   114588 . . . 114386 by Method #2        -   114295 . . . 113851 by Method #2        -   115813 . . . 115448 by Method #3        -   115105 . . . 114911 by Method #3        -   114822 . . . 114700 by Method #3        -   114588 . . . 114386 by Method #3        -   114295 . . . 113337 by Method #3

(Ac) cDNA Sequence

All the gi numbers were assigned by Genbank to track the public genomicsequences except:

gi 1000000001

gi 1000000002

gi 1000000003

gi 1000000004; and

gi 1000000005.

These gi numbers were assigned by Applicants to the five Arabidopsischromosome sequences that were published by the Institute of GenomeResearch (TIGR). Gi 1000000001 corresponds to chromosome 1, Gi1000000002 to chromosome 2, etc.

The method of annotation is indicated as well as any similar publicannotations.

f. Promoters & Terminators

Promoter sequences are 5′ of the translational start site in a gene;more typically, 5′ of the transcriptional start site or sites.Terminator sequences are 3′ of the translational terminator codon; moretypically, 3′ of the end of the 3′ UTR.

For even more specifics of the Reference and Sequence Tables, see thesection below titled “Brief Description of the Tables.”

I.B. Transcriptional (Differential Expression) Information—Introductionto Differential Expression Data & Analyses

A major way that a cell controls its response to internal or externalstimuli is by regulating the rate of transcription of specific genes.For example, the differentiation of cells during organogenensis intoforms characteristic of the organ is associated with the selectiveactivation and repression of large numbers of genes. Thus, specificorgans, tissues and cells are functionally distinct due to the differentpopulations of mRNAs and protein products they possess. Internal signalsprogram the selective activation and repression programs. For example,internally synthesized hormones produce such signals. The level ofhormone can be raised by increasing the level of transcription of genesencoding proteins concerned with hormone synthesis.

To measure how a cell reacts to internal and/or external stimuli,individual mRNA levels can be measured and used as an indicator for theextent of transcription of the gene. Cells can be exposed to a stimulus,and mRNA can be isolated and assayed at different time points afterstimulation. The mRNA from the stimulated cells can be compared tocontrol cells that were not stimulated. The mRNA levels of particularMaximum Length Sequences that are higher in the stimulated cell versusthe control indicate a stimulus-specific response of the cell. The sameis true of mRNA levels that are lower in stimulated cells versus thecontrol condition. Similar studies can be performed with cells takenfrom an organism with a defined mutation in their genome as comparedwith cells without the mutation. Altered mRNA levels in the mutatedcells indicate how the mutation causes transcriptional changes. Thesetranscriptional changes are associated with the phenotype that themutated cells exhibit that is different from the phenotype exhibited bythe control cells.

Applicants have utilized microarray techniques to measure the levels ofmRNAs in cells from mutant plants, stimulated plants, and/or selectedfrom specific organs. The differential expression of various genes inthe samples versus controls are listed in the MA_diff Tables. Applicantshave analyzed the differential data to identify genes whose mRNAtranscription levels are positively correlated. From these analyses,Applicants were able to group different genes together whosetranscription patterns are correlated. The results of the analyses arereported in the MA_clust Tables.

a. Experimental Detail

A microarray is a small solid support, usually the size of a microscopeslide, onto which a number of polynucleotides have been spotted onto orsynthesized in distinct positions on the slide (also referred to as achip). Typically, the polynucleotides are spotted in a grid formation.The polynucleotides can either be Maximum Length Sequences or shortersynthetic oligonucleotides, whose sequence is complementary to specificMaximum Length Sequence entities. A typical chip format is as follows:

Oligo #1 Oligo #2 Oligo #3 Oligo #4 Oligo #5 Oligo #6 Oligo #7 Oligo #8Oligo #9

For Applicants' experiments, samples were hybridized to the chips usingthe “two-color” microarray procedure. A fluorescent dye was used tolabel cDNA reverse-transcribed from mRNA isolated from cells that hadbeen stimulated, mutated, or collected from a specific organ ordevelopmental stage. A second fluorescent dye of another color was usedto label cDNA prepared from control cells.

The two differentially-labeled cDNAs were mixed together. Microarraychips were incubated with this mixture. For Applicants' experiments thetwo dyes that are used are Cy3, which fluoresces in the red color range,and Cy5, which fluoresces in the green/blue color range. Thus, if:

cDNA#1 binds to Oligo #1;

cDNA#1 from the sample is labeled red;

cDNA#1 from the control is labeled green, and

cDNA#1 is in both the sample and control,

then cDNA#1 from both the sample and control will bind to Oligo#1 on thechip. If the sample has 10 times more cDNA#1 than the control, then 10times more of the cDNA#1 would be hybridized to Oligo#1. Thus, the spoton the chip with Oligo#1 spot would look red.

Oligo #2 Oligo #3 Oligo #4 Oligo #5 Oligo #6 Oligo #7 Oligo #8 Oligo #9If the situation were reversed, the spot would appear green. If thesample has approximately the same amount of cDNA#1 as the control, thenthe Oligo#1 spot on the chip would look yellow. These colordifferentials are measured quantitatively and used to deduce therelative concentration of mRNAs from individual genes in particularsamples.

b. MA_Diff Data Table

To generate data, Applicants labeled and hybridized the sample andcontrol mRNA in duplicate experiments. One chip was exposed to a mixtureof cDNAs from both a sample and control, where the sample cDNA waslabeled with Cy3, and the control was labeled with Cy5 dye. For thesecond labeling and chip hybridization experiments, the fluorescentlabels were reversed; that is, the Cy5 dye for the sample, and the Cy3dye for the control.

Whether Cy5 or Cy3 was used to label the sample, the fluorescenceproduced by the sample was divided by the fluorescence of the control. AcDNA was determined to be differentially expressed in response to thestimulus in question if a statistically-significantly ration differencein the sample versus the control was measured by both chip hybridizationexperiments.

The MA_diff tables show which cDNA were significantly up-regulated asdesignated by a “+” and which were significantly down-regulated asdesignated by a “−” for each pair of chips using the same sample andcontrol.

I.C. Phenotypic Information

One means of determining the phenotypic effect of a gene is either toinsert extra active copies of the gene or coding sequence, or to disruptan existing copy of the gene in a cell or organism and measure theeffects of the genetic change on one or more phenotypic characters ortraits. “Knock-in” is used herein to refer to insertion of additionalactive copies of a gene or coding sequence. “Knock-out” refers to aplant where an endogenous gene(s) is disrupted. Applicants have usedboth methods of addition or disruption to determine the phenotypiceffects of gene or gene components or products, and have therebydiscovered the function of the genes and their utilities.

1. Knock-In Results

The coding sequence of a desired protein can be functionally linked to aheterologous promoter to facilitate expression. Here, Applicants haveoperably linked a number of coding sequences to either one of thepromoters listed below:

Specific Promoter Plant Line GFP Pattern activity Descriptor Rootepidermis/mostly toward the lower Specific to the root basal Root basalregion of root (more intense than CS9094) region. Root-endodermis/cortex(initials sharp); Specific to the root Root/Petiole/Flowersshoot-mesophyll of one leaf, sharp guard cell endodermis-cortex marking.New leaf petioles near tip of region, leaf petiole, and primaryinflorescence; floral stems; in flowers. flowers at base of sepal,anther stems, and pistil Broad root exp. (some dermal, some cortical,Specific to root and stem. Root/Stem1 some vascular); shoot apex.Faintly in petiole; stem High expression in stem, excluded from 1stSpecific to stem and root. Root/Stem2 true leaves/High in root. Faintexpression in stem Shoot meristem/whole root region; little bit Specificto roots, shoot Root/Stem/Leaves/Flowers on cotyledons. Base ofleaves(axillary meristem, base of leaves meristem?); base of sepals;inflorescence and flowers. meristem; small amount in unfertilizedpistil. root tip vascular initials; vascular system Specific to vascularVascular/Ovule/Young throughout plant; Bud petal vasculature andsystems. Seed/Embryo pistil septum; Flower petal vascualture; Flowerpistil septum; Pre fertilization ovules; Post fertilization ovule atchalazal end; Developing seed (young, maturing siliques); Seed coat andyoung embryos. GFP not observed in mature embryos. Flower,sepal/vascular tissue of root, stem, Specific to flowers, seedFlowers/Seed/Vasculature/ and cotyledons. Stems of new flowers; andvasculature. Embryo vasculature or petals, anthers, sepals, andpistil/silique; Vasculature throughtout seedling: root, hypocotyl,petioles, stem, cotyledons, first true leaves; Rosette vasculature;Cauline leaf vasculature; Bud pedicel vasculature; Flower vasculature:(sepals, petals, filaments, pistil); Bud vasculature (sepal, petal,filament, pistil); Funiculus in both flower and bud; Some possible seedcoat expression; Silique funiculus; Very faint fluorescence in matureembryo (auto fluorescence perhaps); Root expression - primarily incortex (upper Specific to root. Roots2 refion of the root). No shootexpression Root expression - less intense in whole root Specific to rootand shoot Root/SAM of young seedling. Shoot apical meristem; apicalmeristem. organ primordia in SAM region. Root epidermis/tip; shootepidermis/vascular; Specific to seed and to Seed/Epidermis/Ovary/ leafepidermis; expression in developing epidermal layers of roots, Fruitseed/ovule - mature embryo; Primary and shoots and leaves. lateral rootcortex; Very strong in root cap; Base of flower bud and epidermis ofcarpels; Base of flower, epidermis of filaments, epidermis of carpels;Trichomes; Weak (hardly detectable) gfp expression in vasculaturethroughout seedling; Strong expression in trichomes; POST-fertilizationSEED only; GFP strength increases as silique matures; Weak at suspensorend of the embryo; GFP observed in seed coat; Root and postfertilization seed specific gfp expression; Expression in seed coat.Young root dermis; dermal/cortical?/vascular Specific to roots, shoots,Roots/Shoots/Ovule in older root; general (epidermal?) shoot and ovules.expression; ovules. some in sepals; vasculature of stem Vascular tissueof root; Meristem tissues: Specific to root structuralVasculature/Meristem axillary meristems, floral meristems, base of leafvascular region and flowers/sepals; Weak expression in to floral budsand axillary hypocotyl, petiole and cotyledon meristem vasculature..

The chimeric constructs were transformed into Arabidopsis thaliana. Theresulting transformed lines were screened to determine what phenotypeswere changed due to introduced transgene. The phenotype changes,relative to the control, are reported in the Knock-in tables.

2. Knock-Out Results

Knock-out plants in Arabidopsis thaliana were created by inserting apolynucleotide tag into the genome. The location of the tag wasidentified using primers to the tag sequence and isolation of the plantgenomic sequence that flanks the tag using a variation of the polymerasechain reaction. The plants were generated using the procedure describedin Feldmann et al., (1987) Molec. Gen. Genet. 208: 1-9; Feldmann (1991)Plant Journal, 1:71-83 and Forsthoefel et al., (1992) Aust. J. PlantPhysiol. 19:353-366. On average, the population of plants that wasscreened had ˜1.5 to 2 tags. Generally, the number of tags ranged from 1to greater than 5.

The polynucleotide tags were classified as either incorporated within agene, or between two genes. The data in the Knock-out Table indicateswhich plants have a tag(s) causing a disruption in a gene, or adisruption between genes.

a. Disruption in a Gene

For the sake of this analysis, the tag was considered to be causing adisruption in a gene when the tag was located:

1) less than 501 upstream of the transcriptional start site;

2) less than 701 upstream of the translational initiation codon;

3) between the translational initiation and termination codons of thegene,

4) less than 301 downstream of the translational stop codon; or

5) less than 151 downstream of a transcriptional termination site.

By this definition, a tag can be inserted in two genes. For example, iftwo genes have only 700 nucleotides between the translationaltermination codon of one gene and the translational initiation codon ofthe other gene, the tag can be inserted into the terminator of one geneand the promoter of the other gene according to the definition above.

Genomic annotations by the method OCKHAM-OCDNA identify thetranscriptional start and stop site of a gene.

b. Disruption Between Genes

When a tag causes a disruption between two genes, either or both genescan be affected. Typically, a tag can affect a gene if it disrupts thegenome at a location 3000 nt downstream to the start codon of a gene.More typically, insertions found 1000-2000 nt upstream (5′), or 750-1000nt downstream (3′) could be expected to disrupt expression.

c. More than One Insert

A plant can have multiple tags. If a mutant phenotype is observed, thenit can be attributed to any one or all of the tags.

I.D. Brief Description of the Figures and Individual Tables

Figures

1. FIG. 1 illustrates the Genomics Engine used by Applicants and depictshow gene sequences were determined using five different types oftechnologies.

2. FIG. 2 illustrates how genes are activated by internal stimuli andprotein is produced from them.

3. FIG. 3 illustrates the integration of data across species to linkgene products and phenotypes.

4. FIG. 4 illustrates the regions found in a typical MLS.

5. FIG. 5 is a graph illustrating how the genes, gene components andproducts were classified as either early or late responders following aspecific treatment.

6. FIG. 6 shows expression pattern of a cell wall synthesis gene, cDNAID1595707, during fruit development.

7. FIG. 7 shows the different regions of a typical gene.

Tables 1. Reference and Sequence Tables

The sequences of exemplary SDFs and polypeptides corresponding to thecoding sequences of the instant invention are described in the Referenceand Sequence Tables (sometimes referred to as the REF and SEQ Tables.The Reference Table refers to a number of “Maximum Length Sequences” or“MLS.” Each MLS corresponds to the longest cDNA obtained, either bycloning or by the prediction from genomic sequence. The sequence of theMLS is the cDNA sequence as described in the Av subsection of theReference Table.

The Reference Table includes the following information relating to eachMLS:

I. cDNA Sequence A. 5′ UTR B. Coding Sequence C. 3′ UTR II. GenomicSequence A. Exons B. Introns C. Promoters III. Link of cDNA Sequences toClone IDs IV. Multiple Transcription Start Sites V. PolypeptideSequences A. Signal Peptide B. Domains C. Related Polypeptides VI.Related Polynucleotide Sequences

I. cDNA Sequence

The Reference Table indicates which sequence in the Sequence Tablerepresents the sequence of each MLS. The MLS sequence can comprise 5′and 3′ UTR as well as coding sequences. In addition, specific cDNA clonenumbers also are included in the Reference Table when the MLS sequencerelates to a specific cDNA clone.

A. 5′ UTR

The location of the 5′ UTR can be determined by comparing the most 5′MLS sequence with the corresponding genomic sequence as indicated in theReference Table. The sequence that matches, beginning at any of thetranscriptional start sites and ending at the last nucleotide before anyof the translational start sites corresponds to the 5′ UTR.

B. Coding Region

The coding region is the sequence in any open reading frame found in theMLS. Coding regions of interest are indicated in the PolyP SEQsubsection of the Reference Table.

C. 3′ UTR

The location of the 3′ UTR can be determined by comparing the most 3′MLS sequence with the corresponding genomic sequence as indicated in theReference Table. The sequence that matches, beginning at thetranslational stop site and ending at the last nucleotide of the MLScorresponds to the 3′ UTR.

II. Genomic Sequence

Further, the Reference Table indicates the specific “gi” number of thegenomic sequence if the sequence resides in a public databank. For eachgenomic sequence, Reference tables indicate which regions are includedin the MLS. These regions can include the 5′ and 3′ UTRs as well as thecoding sequence of the MLS. See, for example, the scheme depicted inFIG. 4.

The Reference Table reports the first and last base of each region thatare included in an MLS sequence. An example is shown below:

gi No. 47000:

37102 . . . 37497

37593 . . . 37925

The numbers indicate that the MLS contains the following sequences fromtwo regions of gi No. 47000; a first region including bases 37102-37497,and a second region including bases 37593-37925.

A. Exon Sequences

The location of the exons can be determined by comparing the sequence ofthe regions from the genomic sequences with the corresponding MLSsequence as indicated by the Reference Table.

i. Initial Exon

To determine the location of the initial exon, information from the

(1) polypeptide sequence section;

(2) cDNA polynucleotide section; and

(3) the genomic sequence section

of the Reference Table is used. First, the polypeptide section willindicate where the translational start site is located in the MLSsequence. The MLS sequence can be matched to the genomic sequence thatcorresponds to the MLS. Based on the match between the MLS andcorresponding genomic sequences, the location of the translational startsite can be determined in one of the regions of the genomic sequence.The location of this translational start site is the start of the firstexon.

Generally, the last base of the exon of the corresponding genomicregion, in which the translational start site was located, willrepresent the end of the initial exon. In some cases, the initial exonwill end with a stop codon, when the initial exon is the only exon.

In the case when sequences representing the MLS are in the positivestrand of the corresponding genomic sequence, the last base will be alarger number than the first base. When the sequences representing theMLS are in the negative strand of the corresponding genomic sequence,then the last base will be a smaller number than the first base.

ii. Internal Exons

Except for the regions that comprise the 5′ and 3′ UTRs, initial exon,and terminal exon, the remaining genomic regions that match the MLSsequence are the internal exons. Specifically, the bases defining theboundaries of the remaining regions also define the intron/exonjunctions of the internal exons.

iii. Terminal Exon

As with the initial exon, the location of the terminal exon isdetermined with information from the

(1) polypeptide sequence section;

(2) cDNA polynucleotide section; and

(3) the genomic sequence section

of the Reference Table. The polypeptide section will indicate where thestop codon is located in the MLS sequence. The MLS sequence can bematched to the corresponding genomic sequence. Based on the matchbetween MLS and corresponding genomic sequences, the location of thestop codon can be determined in one of the regions of the genomicsequence. The location of this stop codon is the end of the terminalexon. Generally, the first base of the exon of the corresponding genomicregion that matches the cDNA sequence, in which the stop codon waslocated, will represent the beginning of the terminal exon. In somecases, the translational start site will represent the start of theterminal exon, which will be the only exon.

In the case when the MLS sequences are in the positive strand of thecorresponding genomic sequence, the last base will be a larger numberthan the first base. When the MLS sequences are in the negative strandof the corresponding genomic sequence, then the last base will be asmaller number than the first base.

B. Intron Sequences

In addition, the introns corresponding to the MLS are defined byidentifying the genomic sequence located between the regions where thegenomic sequence comprises exons. Thus, introns are defined as startingone base downstream of a genomic region comprising an exon, and end onebase upstream from a genomic region comprising an exon.

C. Promoter Sequences

As indicated below, promoter sequences corresponding to the MLS aredefined as sequences upstream of the first exon; more usually, assequences upstream of the first of multiple transcription start sites;even more usually as sequences about 2,000 nucleotides upstream of thefirst of multiple transcription start sites.

III. Link of cDNA Sequences to Clone IDs

As noted above, the Reference Table identifies the cDNA clone(s) thatrelate to each MLS. The MLS sequence can be longer than the sequencesincluded in the cDNA clones. In such a case, the Reference Tableindicates the region of the MLS that is included in the clone. If eitherthe 5′ or 3′ termini of the cDNA clone sequence is the same as the MLSsequence, no mention will be made.

IV. Multiple Transcription Start Sites

Initiation of transcription can occur at a number of sites of the gene.The Reference Table indicates the possible multiple transcription sitesfor each gene. In the Reference Table, the location of the transcriptionstart sites can be either a positive or negative number.

The positions indicated by positive numbers refer to the transcriptionstart sites as located in the MLS sequence. The negative numbersindicate the transcription start site within the genomic sequence thatcorresponds to the MLS.

To determine the location of the transcription start sites with thenegative numbers, the MLS sequence is aligned with the correspondinggenomic sequence. In the instances when a public genomic sequence isreferenced, the relevant corresponding genomic sequence can be found bydirect reference to the nucleotide sequence indicated by the “gi” numbershown in the public genomic DNA section of the Reference Table. When theposition is a negative number, the transcription start site is locatedin the corresponding genomic sequence upstream of the base that matchesthe beginning of the MLS sequence in the alignment. The negative numberis relative to the first base of the MLS sequence which matches thegenomic sequence corresponding to the relevant “gi” number.

In the instances when no public genomic DNA is referenced, the relevantnucleotide sequence for alignment is the nucleotide sequence associatedwith the amino acid sequence designated by “gi” number of the laterPolyP SEQ subsection.

V. Polypeptide Sequences

The PolyP SEQ subsection lists SEQ ID NOs and Ceres SEQ ID NO forpolypeptide sequences corresponding to the coding sequence of the MLSsequence and the location of the translational start site with thecoding sequence of the MLS sequence.

The MLS sequence can have multiple translational start sites and can becapable of producing more than one polypeptide sequence.

A. Signal Peptide

The Reference tables also indicate in subsection (B) the cleavage siteof the putative signal peptide of the polypeptide corresponding to thecoding sequence of the MLS sequence. Typically, signal peptide codingsequences comprise a sequence encoding the first residue of thepolypeptide to the cleavage site residue.

B. Domains

Subsection (C) provides information regarding identified domains (wherepresent) within the polypeptide and (where present) a name for thepolypeptide domain.

C. Related Polypeptides

Subsection (Dp) provides (where present) information concerning aminoacid sequences that are found to be related and have some percentage ofsequence identity to the polypeptide sequences of the Reference andSequence Tables. These related sequences are identified by a “gi”number.

VI. Related Polynucleotide Sequences

Subsection (Dn) provides polynucleotide sequences (where present) thatare related to and have some percentage of sequence identity to the MLSor corresponding genomic sequence.

Abbreviation Description Max Len. Seq. Maximum Length Sequence rel toRelated to Clone Ids Clone ID numbers Pub gDNA Public Genomic DNA gi No.gi number Gen. Seq. in Cdna Genomic Sequence in cDNA (Each region for asingle gene prediction is listed on a separate line. In the case ofmultiple gene predictions, the group of regions relating to a singleprediction are separated by a blank line) (Ac) cDNA SEQ cDNA sequencePat. Appln. SEQ ID NO Patent Application SEQ ID NO: Ceres SEQ ID CeresSEQ ID NO: NO: 1673877 SEQ # w. TSS Location within the cDNA sequence,SEQ ID NO:, of Transcription Start Sites which are listed below Clone ID#: # -> # Clone ID comprises bases # to # of the cDNA Sequence PolyP SEQPolypeptide Sequence Pat. Appln. SEQ ID NO: Patent Application SEQ IDNO: Ceres SEQ ID NO Ceres SEQ ID NO: Loc. SEQ ID NO: @ nt. Location oftranslational start site in cDNA of SEQ ID NO: at nucleotide number (C)Pred. PP Nom. & Nomination and Annotation of Domains within Annot.Predicted Polypeptide(s) (Title) Name of Domain Loc. SEQ ID Location ofthe domain within the polypeptide NO #: # -> # aa. of SEQ ID NO: from #to # amino acid residues. (Dp) Rel. AA SEQ Related Amino Acid SequencesAlign. NO Alignment number gi No Gi number Desp. Description % Idnt.Percent identity Align. Len. Alignment Length Loc. SEQ ID Locationwithin SEQ ID NO: from # to # NO: # -> # aa amino acid residue.

2. Protein Group Table

This table indicates groups of proteins that share a signature sequence(also referred to as a consensus sequence). The Protein group alsoreferred to as the Ortholog group is named by the peptide ID with whichall members were compared. Each group contains sequences that wereincluded at the 10⁻⁵⁰, 10⁻³⁰, and 10⁻¹⁰ p-value cutoffs. For each group,the peptide ID and at which cutoff the peptide was included into thegroup. The same peptide ID may be included in the group three times aspeptide ID 50, peptide ID 30 and peptide ID 10. The data indicates thatpeptide ID was included in the group when the threshold was either10⁻⁵⁰, 10⁻³⁰, or 10⁻¹⁰. All the peptide IDs that are followed by “50”were included in the protein group when the e-value cutoff was 10⁻⁵⁰.All the peptide IDs that are followed by either “30” or “50” wereincluded in the protein group when the threshold e-value was 10⁻³⁰. Allthe peptide IDs that are followed by “10”, “30” or “50” were included inthe protein group when 10⁻¹⁰ was used as the e-value cutoff. At the endof each protein group is a list of the consensus sequence that proteinsshare at the 10⁻⁵⁰, 10⁻³⁰, or 10⁻¹⁰. The consensus sequence containsboth lower-case and upper-case letters. The upper-case letters representthe standard one-letter amino acid abbreviations. The lower case lettersrepresent classes of amino acids:

-   -   “t” refers to tiny amino acids, which are specifically alanine,        glycine, serine and threonine.    -   “p” refers to polar amino acids, which are specifically,        asparagine and glutamine    -   “n” refers to negatively charged amino acids, which are        specifically, aspartic acid and glutamic acid    -   “+” refers to positively charged residues, which are        specifically, lysine, arginine, and histidine    -   “r” refers to aromatic residues, which are specifically,        phenylalanine, tyrosine, and tryptophan,    -   “a” refers to aliphatic residues, which are specifically,        isoleucine, valine, leucine, and methonine

3. Protein Group_Matrix Table

In addition to each consensus sequence, Applicants have generated ascoring matrix to provide further description of the consensus sequence.The first row of each matrix indicates the residue position in theconsensus sequence. The matrix reports number of occurrences of all theamino acids that were found in the group members for every residueposition of the signature sequence. The matrix also indicates for eachresidue position, how many different organisms were found to have apolypeptide in the group that included a residue at the relevantposition. The last line of the matrix indicates all the amino acids thatwere found at each position of the consensus.

4. MA_diff Table

The MA_diff Table presents the results of the differential expressionexperiments for the mRNAs, as reported by their corresponding cDNA IDnumber, that were differentially transcribed under a particular set ofconditions as compared to a control sample. The cDNA ID numberscorrespond to those utilized in the Reference and Sequence Tables.Increases in mRNA abundance levels in experimental plants versus thecontrols are denoted with the plus sign (+). Likewise, reductions inmRNA abundance levels in the experimental plants are denoted with theminus (−) sign.

The Table is organized according to each set of experimental conditions,which are denoted by the term “Expt ID:” followed by a particularnumber. The table below links each Expt ID with a short description ofthe experiment and the parameters.

For each experiment ID a method of the normalization is specified.“Method: 2” represents normalization by median the goal of the method isto adjust the ratios by a factor so that the median of the ratiodistribution is 1. Method 3 is the normalization procedure conducted byAglilent Technologies, Inc. Palo Alto, Calif., USA.

The MA_diff Table also specifies the specific parameters and theexperiment number (e.g. 107871) used in compiling the data. Theexperiment numbers are referenced in the appropriate utility/functionssections herein. The background threshold was set to “BKG_Threshold=X”to reduce the effect of the background on the signal.

Finally, the Table includes reference to an “Organism_ID” number. Thisnumber refers to the cDNA spotted on the chip were similar toArabidopsis thaliana (3769) sequences or whether the oligo used for thechips were similar to Zea mays (311987) sequences.

5. MA_diff (Experiment) Table

The following Table summarizes the experimental procedures utilized forthe differential expression experiments, each experiment beingidentified by a unique “Expt ID” number.

Exam- Experiment ple No. short name genome EXPT_ID Value PARAMETER UNITS3ii 3642-1 Arabidopsis 108512 3746-1 Plant Line Hours 3nArab_0.001%_MeJA_1 Arabidopsis 108568 Aerial Tissue Tissue 0.001%_MeJATreatment Compound 1 Timepoint Hours 3n Arab_0.001%_MeJA_1 Arabidopsis108569 Aerial Tissue Tissue 6 Timepoint Hours 0.001%_MeJA TreatmentCompound 3j Arab_0.1uM_Epi- Arabidopsis 108580 Aerial Tissue TissueBrass_1 1 Timepoint Hours 0.1uM_Brassino_Steroid Treatment Compound 3jArab_0.1uM_Epi- Arabidopsis 108581 Aerial Tissue Tissue Brass_1 6Timepoint Hours 0.1uM_Brassino_Steroid Treatment Compound 3gArab_100uM_ABA_1 Arabidopsis 108560 Aerial Tissue Tissue 1 TimepointHours 100uM_ABA Treatment Compound 3g Arab_100uM_ABA_1 Arabidopsis108561 Aerial Tissue Tissue 100uM_ABA Treatment Compound 6 TimepointHours 3I Arab_100uM_BA_1 Arabidopsis 108566 Aerial Tissue Tissue 1Timepoint Hours 100uM_BA Treatment Compound 3I Arab_100uM_BA_1Arabidopsis 108567 Aerial Tissue Tissue 100uM_BA Treatment Compound 6Timepoint Hours 3k Arab_100uM_GA3_1 Arabidopsis 108562 Aerial TissueTissue 1 Timepoint Hours 100 uM GA3 Treatment Compound 3kArab_100uM_GA3_1 Arabidopsis 108563 Aerial Tissue Tissue 100 uM GA3Treatment Compound 6 Timepoint Hours 3h Arab_100uM_NAA_1 Arabidopsis108564 Aerial Tissue Tissue 1 Timepoint Hours 100uM_NAA TreatmentCompound 3h Arab_100uM_NAA_1 Arabidopsis 108565 Aerial Tissue Tissue100uM_NAA Treatment Compound 6 Timepoint Hours 3r Arab_20%_PEG_1Arabidopsis 108570 Aerial Tissue Tissue 1 Timepoint Hours 20% PEGTreatment Compound 3r Arab_20%_PEG_1 Arabidopsis 108571 Aerial TissueTissue 20% PEG Treatment Compound 6 Timepoint Hours 3o Arab_2mM_SA_1Arabidopsis 108586 Aerial Tissue Tissue 2mM_SA Treatment Compound 1Timepoint Hours 3o Arab_2mM_SA_1 Arabidopsis 108587 Aerial Tissue Tissue6 Timepoint Hours 2mM_SA Treatment Compound 3u Arab_5mM_H2O2_1Arabidopsis 108582 Aerial Tissue Tissue 1 Timepoint Hours 5mM_H2O2Treatment Compound 3u Arab_5mM_H2O2_1 Arabidopsis 108583 Aerial TissueTissue 5mM_H2O2 Treatment Compound 6 Timepoint Hours 3v Arab_5mM_NaNP_1Arabidopsis 108584 Aerial Tissue Tissue 1 Timepoint Hours 5mM_NaNPTreatment Compound 3v Arab_5mM_NaNP_1 Arabidopsis 108585 Aerial TissueTissue 5mM_NaNP Treatment Compound 6 Timepoint Hours 3t Arab_Cold_1Arabidopsis 108578 Aerial Tissue Tissue Cold Treatment Compound 1Timepoint Hours 3t Arab_Cold_1 Arabidopsis 108579 Aerial Tissue Tissue 6Timepoint Hours Cold Treatment Compound 3g Arab_Drought_1 Arabidopsis108572 Aerial Tissue Tissue 1 Timepoint Hours Drought Treatment Compound3g Arab_Drought_1 Arabidopsis 108573 Aerial Tissue Tissue DroughtTreatment Compound 6 Timepoint Hours 3s Arab_Heat_1 Arabidopsis 108576Aerial Tissue Tissue 1 Timepoint Hours Heat (42 deg Treatment CompoundC.) 3s Arab_Heat_1 Arabidopsis 108577 Aerial Tissue Tissue Heat (42 degTreatment Compound C.) 6 Timepoint Hours 3aa Arab_Ler- Arabidopsis108595 Ler_pi Plant Line Hours (ovule) pi_ovule_1 Ovule Tissue Tissue 3bArab_Ler- Arabidopsis 108594 Ler_rhl Plant Line Hours rhl_root_1 RootTissue Tissue 3l Arab_NO3_H- Arabidopsis 108592 Aerial Tissue Tissueto-L_1 Low Nitrogen Treatment Compound 12 Timepoint Hours 3l Arab_NO3_H-Arabidopsis 108593 Aerial Tissue Tissue to-L_1 24 Timepoint Hours LowNitrogen Treatment Compound 3l Arab_NO3_L- Arabidopsis 108588 AerialTissue Tissue to-H_1 2 Timepoint Hours Nitrogen Treatment Compound 3lArab_NO3_L- Arabidopsis 108589 Aerial Tissue Tissue to-H_1 NitrogenTreatment Compound 6 Timepoint Hours 3l Arab_NO3_L- Arabidopsis 108590Aerial Tissue Tissue to-H_1 9 Timepoint Hours Nitrogen TreatmentCompound 3l Arab_NO3_L- Arabidopsis 108591 Aerial Tissue Tissue to-H_1Nitrogen Treatment Compound 12 Timepoint Hours 3p Arab_Wounding_1Arabidopsis 108574 Aerial Tissue Tissue 1 Timepoint Hours WoundingTreatment Compound 3p Arab_Wounding_1 Arabidopsis 108575 Aerial TissueTissue Wounding Treatment Compound 6 Timepoint Hours 3o Columbia/CS3726Arabidopsis 108475 Columbia species Hours flower SA SA TreatmentCompound 5 weeks Timepoint Hours 3o Columbia/CS3726 Arabidopsis 108476CS3726 species Hours flower SA 5 weeks Timepoint Hours SA TreatmentCompound 3p Corn_0.001Percent_MeJA Zea Mays 108555 Aerial Tissue Tissue24 Timepoint Hours 0.001%_MeJA Treatment Compound 3jCorn_0.1uM_Brassino_Steroid Zea Mays 108557 24 Timepoint Hours AerialTissue Tissue 0.1uM_Brassino_Steroid Treatment Compound 3gCorn_100uM_ABA Zea Mays 108513 Aerial Tissue Tissue ABA TreatmentCompound 6 Timepoint Hours 3g Corn_100uM_ABA Zea Mays 108597 AerialTissue Tissue 24 Timepoint Hours 100uM_ABA Treatment Compound 3iCorn_100uM_BA Zea Mays 108517 Aerial Tissue Tissue 6 Timepoint Hours BATreatment Compound 3k Corn_100uM_GA3 Zea Mays 108519 Aerial TissueTissue 100 uM Treatment Compound Giberillic Acid 1 Timepoint Hours 3kCorn_100uM_GA3 Zea Mays 108520 Aerial Tissue Tissue 6 Timepoint Hours100 uM Treatment Compound Giberillic Acid 3k Corn_100uM_GA3 Zea Mays108521 Aerial Tissue Tissue 100 uM Treatment Compound Giberillic Acid 12Timepoint Hours 3h Corn_100uM_NAA Zea Mays 108516 Aerial Tissue TissueNAA Treatment Compound 6 Timepoint Hours 3h Corn_100uM_NAA Zea Mays108554 Aerial Tissue Tissue 24 Timepoint Hours NAA Treatment Compound3hh Corn_1400- Zea Mays 108598 Shoot apices Tissue Tissue 6/S-17 3rCorn_150mM_NaCl Zea Mays 108541 Aerial Tissue Tissue 1 Timepoint Hours150mM_NaCl Treatment Compound 3r Corn_150mM_NaCl Zea Mays 108542 AerialTissue Tissue 150mM_NaCl Treatment Compound 6 Timepoint Hours 3rCorn_150mM_NaCl Zea Mays 108553 Aerial Tissue Tissue 24 Timepoint Hours150mM_NaCl Treatment Compound 3r Corn_20%_PEG Zea Mays 108539 AerialTissue Tissue 1 Timepoint Hours 20% PEG Treatment Compound 3rCorn_20%_PEG Zea Mays 108540 Aerial Tissue Tissue 20% PEG TreatmentCompound 6 Timepoint Hours 3o Corn_2mM_SA Zea Mays 108515 Aerial TissueTissue SA Treatment Compound 12 Timepoint Hours 3o Corn_2mM_SA Zea Mays108552 Aerial Tissue Tissue SA Treatment Compound 24 Timepoint Hours 3uCorn_5mM_H2O2 Zea Mays 108537 Aerial Tissue Tissue H2O2 TreatmentCompound 1 Timepoint Hours 3u Corn_5mM_H2O2 Zea Mays 108538 AerialTissue Tissue 6 Timepoint Hours H2O2 Treatment Compound 3u Corn_5mM_H2O2Zea Mays 108558 Aerial Tissue Tissue 24 Timepoint Hours H2O2 TreatmentCompound 3v Corn_5mM_NO Zea Mays 108526 Aerial Tissue Tissue NOTreatment Compound 1 Timepoint Hours 3v Corn_5mM_NO Zea Mays 108527Aerial Tissue Tissue 6 Timepoint Hours NO Treatment Compound 3vCorn_5mM_NO Zea Mays 108559 Aerial Tissue Tissue 12 Timepoint Hours NOTreatment Compound 3t Corn_Cold Zea Mays 108533 Aerial Tissue Tissue 1Timepoint Hours Cold Treatment Compound 3t Corn_Cold Zea Mays 108534Aerial Tissue Tissue Cold Treatment Compound 6 Timepoint Hours 3qCorn_Drought Zea Mays 108502 Drought Treatment Compound 1 TimepointHours 3q Corn_Drought Zea Mays 108503 Drought Treatment Compound 6Timepoint Hours 3q Corn_Drought Zea Mays 108504 Drought TreatmentCompound 12 Timepoint Hours 3q Corn_Drought Zea Mays 108556 DroughtTreatment Compound 24 Timepoint Hours 3s Corn_Heat Zea Mays 108522Aerial Tissue Tissue 1 Timepoint Hours Heat (42 deg Treatment CompoundC.) 3s Corn_Heat Zea Mays 108523 Aerial Tissue Tissue 6 Timepoint HoursHeat (42 deg Treatment Compound C.) 3gg Corn_Imbibed Zea Mays 108518Imbibed Treatment Compound Seeds 4 Age days old Roots Tissue Tissue 3ggCorn_Imbibed Zea Mays 108528 Imbibed Treatment Compound Seeds AerialTissue Tissue 5 Age days old 3gg Corn_Imbibed Zea Mays 108529 ImbibedTreatment Compound Seeds 5 Age days old Root Tissue Tissue 3ggCorn_Imbibed Zea Mays 108530 Imbibed Treatment Compound Seeds AerialTissue Tissue 6 Age days old 3gg Corn_Imbibed Zea Mays 108531 ImbibedTreatment Compound Seeds 6 Age days old root Tissue Tissue 3ggCorn_Imbibed Zea Mays 108545 Imbibed Treatment Compound Seeds AerialTissue Tissue 3 Age days old 3gg Corn_Imbibed Zea Mays 108546 ImbibedTreatment Compound Seeds 3 Age days old Root Tissue Tissue 3ggCorn_Imbibed Zea Mays 108547 Imbibed Treatment Compound Seeds AerialTissue Tissue 4 Age days old 3gg Corn_Imbibed_Embryo_Endosperm Zea Mays108543 2 Age days old Imbibed Treatment Compound Embryo Tissue Tissue3gg Corn_Imbibed_Embryo_Endosperm Zea Mays 108544 2 Age days oldEndosperm Tissue Tissue Imbibed Treatment Compound 3ee Corn_Meristem ZeaMays 108535 Root Tissue Tissue Meristem 192 Timepoint Hours 3eeCorn_Meristem Zea Mays 108536 Shoot Tissue Tissue Meristem 192 TimepointHours 3n Corn_Nitrogen_H_to_L Zea Mays 108532 Roots Tissue Tissue LowNitrogen Treatment Compound 16 Timepoint Hours 3n Corn_Nitrogen_H_to_LZea Mays 108548 Root Tissue Tissue Low Nitrogen Treatment Compound 4Timepoint Hours 3m Corn_Nitrogen_L_to_H Zea Mays 108549 Aerial TissueTissue 0.166 Timepoint Hours Nitrogen Treatment Compound 3mCorn_Nitrogen_L_to_H Zea Mays 108550 Aerial Tissue Tissue NitrogenTreatment Compound 1.5 Timepoint Hours 3m Corn_Nitrogen_L_to_H Zea Mays108551 Aerial Tissue Tissue 3 Timepoint Hours Nitrogen TreatmentCompound 3ff Corn_RT1 Zea Mays 108599 Unknown Plant Line Hours RootTissue Tissue 3p Corn_Wounding Zea Mays 108524 Aerial Tissue TissueWounding Treatment Compound 1 Timepoint Hours 3p Corn_Wounding Zea Mays108525 Aerial Tissue Tissue 6 Timepoint Hours Wounding TreatmentCompound 3g Drought_Flowers Arabidopsis 108473 Flowers Tissue Tissue 7 dTimepoint Hours Drought Treatment Compound 3g Drought_FlowersArabidopsis 108474 Flowers Tissue Tissue Drought Treatment Compound 8 d(1 d- Timepoint Hours post_re- watering) 3k GA Treated Arabidopsis108484 1 Timepoint Hours 1 Timepoint Hours 3k GA Treated Arabidopsis108485 6 Timepoint Hours 6 Timepoint Hours 3k GA Treated Arabidopsis108486 12 Timepoint Hours 12 Timepoint Hours 3e Germinating Arabidopsis108461 Day 1 Timepoint Hours Seeds 3e Germinating Arabidopsis 108462 Day2 Timepoint Hours Seeds 3e Germinating Arabidopsis 108463 Day 3Timepoint Hours Seeds 3e Germinating Arabidopsis 108464 Day 4 TimepointHours Seeds 3bb Herbicide V3.1 Arabidopsis 108465 Round up TreatmentCompound 12 Timepoint Hours 3bb Herbicide V3.1 Arabidopsis 108466 TrimecTreatment Compound 12 Timepoint Hours 3bb Herbicide V3.1 Arabidopsis108467 Finale Treatment Compound 12 Timepoint Hours 3bb Herbicide V3.1Arabidopsis 108468 Glean Treatment Compound 12 Timepoint Hours 3bbHerbicide_v2 Arabidopsis 107871 Finale Treatment Compound 4 TimepointHours 3bb Herbicide_v2 Arabidopsis 107876 Finale Treatment Compound 12Timepoint Hours 3bb Herbicide_v2 Arabidopsis 107881 Glean TreatmentCompound 4 Timepoint Hours 3bb Herbicide_v2 Arabidopsis 107886 TrimecTreatment Compound 4 Timepoint Hours 3bb Herbicide_v2 Arabidopsis 107891Trimec Treatment Compound 12 Timepoint Hours 3bb Herbicide_v2Arabidopsis 107896 Round-up Treatment Compound 4 Timepoint Hours 3dTrichome Arabidopsis 108452 Hairy Tissue Tissue InflorescencesInfluorescence expt #1 3o SA treatment_1 Arabidopsis 108471 ColumbiaSpecies Hours hour 1 Timepoint Hours SA Treatment Compound 3o SAtreatment_1 Arabidopsis 108472 CS3726 Species Hours hour 1 TimepointHours SA Treatment Compound 3o SA treatment_4 Arabidopsis 108469columbia Species Hours hour 4 Timepoint Hours SA Treatment Compound 3oSA treatment_4 Arabidopsis 108470 CS3726 Species Hours hour SA TreatmentCompound 4 Timepoint Hours 3o SA Arabidopsis 107953 50 Probe % oftreatment_AJ Amount Standard Amount SA Treatment Compound 24 TimepointHours Clontech Probe Type Probe method 3o SA Arabidopsis 107960 50 Probe% of treatment_AJ Amount Standard Amount SA Treatment Compound 24Timepoint Hours Operon Probe Type Probe method 3o SA_treatmentArabidopsis 108443 SA Treatment Compound 24 hour 24 Timepoint Hours 3oSA_treatment 6 Arabidopsis 108440 SA treatment Treatment Compound hour 6hour CS3726 species Hours 3o SA_treatment 6 Arabidopsis 108441 SAtreatment Treatment Compound hour 6 hour Columbia species Hours 3lNitrogen High Arabidopsis 108454 10 min Timepoint Hours transition toLow 3l Nitrogen High Arabidopsis 108455 1 hr Timepoint Hours transitionto Low 3j BR_Shoot Arabidopsis 108478 dwf4-1 Plant Line Hours ApicesExpt 3j BR_Shoot Arabidopsis 108479 AOD4-4 Plant Line Hours Apices Expt3j BR_Shoot Arabidopsis 108480 Ws-2 Plant Line Hours Apices Expt BLTreatment Compound 3j BR_Shoot Arabidopsis 108481 Ws-2 Plant Line HoursApices Expt BRZ Treatment Compound 3jj Tissue Specific Arabidopsis108429 green flower Tissue Tissue Expression operon Probe Type Probemethod 50 Probe % of Amount Standard Amount 3jj Tissue SpecificArabidopsis 108430 white flower Tissue Tissue Expression 50 Probe % ofAmount Standard Amount operon Probe Type Probe method 3jj TissueSpecific Arabidopsis 108431 flowers (bud) Tissue Tissue Expressionoperon Probe Type Probe method 50 Probe % of Amount Standard Amount 3cTissue Specific Arabidopsis 108436 5-10 mm Tissue Tissue Expressionsiliques 33 Probe % of Amount Standard Amount operon Probe Type Probemethod 3c Tissue Specific Arabidopsis 108437 <5 mm Tissue TissueExpression siliques operon Probe Type Probe method 33 Probe % of AmountStandard Amount 3c Tissue Specific Arabidopsis 108438 5 wk siliquesTissue Tissue Expression 33 Probe % of Amount Standard Amount operonProbe Type Probe method 3a Tissue Specific Arabidopsis 108439 Roots (2wk) Tissue Tissue Expression operon Probe Type Probe method 33 Probe %of Amount Standard Amount 3c Tissue Specific Arabidopsis 108497 3 weekTissue Tissue Expression Rossette leaves 100 Probe % of Amount StandardAmount operon Probe Type Probe method 3c Tissue Specific Arabidopsis108498 3-week stems Tissue Tissue Expression operon Probe Type Probemethod 100 Probe % of Amount Standard Amount 3dd U.A.E. Arabidopsis108451 13B12 Plant Line Hours Knockout 3q Ws Arabidopsis Arabidopsis108477 stems and Tissue Tissue Drought 2 days leaves 2 days TimepointHours 3q Ws Arabidopsis Arabidopsis 108482 4 days Timepoint HoursDrought 4 days 3q Ws Arabidopsis Arabidopsis 108483 6 days TimepointHours Drought 6 days 3cc ap2-floral buds Arabidopsis 108501 ap2 (Ler.)Plant Line Hours floral buds Tissue Tissue 3m nitrogen-seed Arabidopsis108487 0.5 Timepoint Hours set 3m nitrogen-seed Arabidopsis 108488 2Timepoint Hours set 3m nitrogen-seed Arabidopsis 108489 4 TimepointHours set 3b rhl mutant2 Arabidopsis 108433 mutant Tissue Tissue 3eeroot tips Arabidopsis 108434 root tips Tissue Tissue 3f stm mutantsArabidopsis 108435 stem Tissue Tissue Aluminum SMD 7304, SMD 7305 AxelSMD 6654, SMD 6655 Cadium SMD 7427, SMD 7428 Cauliflower SMD 5329, SMD5330 Chloroplast SMD 8093, SMD 8094 Circadian SMD 2344, SMD 2359, SMD2361, SMD 2362, SMD 2363, SMD 2364, SMD 2365, SMD 2366, SMD 2367, SMD2368, SMD 3242 CO2 SMD7561, SMD 7562, SMD 7261, SMD 7263, SMD 3710, SMD4649, SMD 4650 Disease SMD 7342, SMD 7343 reactive oxygen SMD 7523 IronSMD 7114, SMD 7115, SMD 7125 defense SMD 8031, SMD 8032 Mitchondria- SMDElectron 8061, Transport SMD 8063 NAA SMD 3743, SMD 3749, SMD 6338, SMD6339 Nitrogen SMD 3787, SMD 3789 Phototropism SMD 4188, SMD 6617, SMD6619 Shade SMD 8130, SMD 7230 Sqn SMD 7133, SMD 7137 Sulfur SMD 8034,SMD 8035 Wounding SMD 3714, SMD 3715 Zinc SMD 7310, SMD 7311

6. MA_Clusters Table

Microarray data was clustered using one of two methods: “completelinkage” or “nearest neighbor” analysis. These clustering methods aredescribed in more detail elsewhere herein. The results of the clusteringanalysis are presented in the MA_clust table. The table is organized asfollows:

“METHOD” refers to a method number which clustering method used.

“CL_METHOD_TYPE=TRUE” refers to complete linkage method.

“NN_METHOD_TYPE=TRUE” refers to the nearest neighbor method.

“FULL_NN_METHOD_TYPE=TRUE” refers to the nearest neighbor method, whereno size limitation was placed on the cluster.

“PARAMETERS” refers to the parameters utilized for the analysis. Thenature of these is also described in more detail elsewhere herein.

“ORGANISM” refers to the cDNA spotted on the chip were similar toArabidopsis thaliana (3769) sequences or whether the oligo used for thechips were similar to Zea mays (311987) sequences.

Each cluster or group of cDNA is identified by a “Group #”, followingwhich are the individual cDNA_Ids that are a member of that Group

7. Knock-In Table

The Knock-In Table presents the results of knock-in experiments whereinplants are grown from tissues transformed with a marker gene-containinginsert and phenotypes are ascertained from the transformed plants. Eachsection of the Table relating to information on a new transformantbegins with a heading “Knock-in phenotype in gene (cDNA_id):” followedby a number which represents the Ceres internal code for a proprietarycDNA sequence. The described transformant was prepared by proceduresdescribed herein, wherein the identified Ceres proprietary cDNA_id(corresponding to the cDNA_id in the Reference and Sequence Tables) wasinterrupted by the marker gene-containing insert. The followinginformation is presented for each section.

-   -   Parent plants used in cross—presents the id numbers of the        parent plants which were crossed to produce the F1 generation        plant for which a phenotype is described. The parent plant with        the promoter is described by a plant line descriptor.    -   Clone ID—presents the clone number of the Ceres proprietary        clone which was the source of the cDNA_id.    -   Phenotype ID—represents an internal identification code.    -   Unique FI plant ID—represents the internal code for the F1 plant        for which a phenotype is described.    -   Assay—presents the type of growth analyzed (e.g. soil gross        morphology), followed by the assay name which corresponds to the        type/location of the tissue that was observed, the name of the        assay conducted for which the result provided the identified        phenotype.    -   Phenotype—describes the phenotype noted for the F1 generation        transformant.    -   Notes—may provide additional information on the described        phenotype for the transform ant.

Each knock-in representing a transformant with an interruption in theidentified cDNA id may be correlated with more than one identifiedphenotype.

8. Knock-Out Table

The Knock-Out Table presents the results of knock-out experimentswherein plants are grown from tissues transformed with a markergene-containing insert wherein phenotypes are ascertained from thetransformed plants. Each section of the Table relating to information ona new transformant begins with a heading “tail id:” representing aninternal code. The following information is presented for each section.

-   -   br—provides another internal code for the experiment.    -   Phenotype_id—provides an identification number for the        particular phenotype identified for the transformant.    -   assay—identifies the assay procedure utilized in the experiment        to identify a phenotype for the transformant.    -   phenotype—represents an internatl identification code.    -   ratio—represents a segregation ratio.    -   notes—lists any notes relevant to the identified phenotype.    -   Knock-out in-genes—Identifies the genes in which the tag has        inserted        -   6) the less than 501 upstream of the transcriptional start            site;        -   7) less than 701 upstream of the translational initiation            codon;        -   8) between the translational initiation and termination            codons of the gene,        -   9) less than 301 downstream of the translational stop codon;            or        -   10) less than 151 downstream of a transcriptional            termination site or a gene.    -   In this table the gene is identified by its cDNA ID number, the        Ceres SEQ ID that is indicated in the (Ac) portion of the        Reference tables. For each cDNA_id, the following information is        provided:        -   the cDNA-id number.        -   in parenthesis, the cluster number of which the identified            cDNA is a member.        -   the “gDNA_Insert pos” representing the position of the            insert in the corresponding gDNA sequence        -   the gi number refers to the TIGR chromosome sequences for            Arabidopsis.    -   Knock-out out of-genes: Identifies the Ceres cDNA proprietary        sequences (noted by cDNA_id which are the same as those        identified in the Reference and Sequence Tables) which are        closest in position to the insert, both upstream and downstream        from the insert. For each cDNA_id, the following information is        provided:        -   In the first parentheses, R indicates that the gene is to            the right of the tag, L indicates that the gene is to right            of the tag as the sequences is read left to right        -   the cDNA_id number        -   in next parentheses, the cluster number of which the            identified cDNA is a member.        -   the distance (in number of nucleotides) of the insert is            upstream of the start of the gene annotation as described in            the Reference Tables or downstream at the end the gene            annotation.        -   the “gDNA_Insert pos” representing the position of the            insert in the corresponding gDNA sequence        -   the gi number refers to the TIGR chromosome sequences for            Arabidopsis.

9. Protein Domain Table

The Protein Domain table provides details concerning the protein domainsnoted in the Reference Table. The majority of the protein domaindescriptions given in the Protein Domain Table are obtained from Prosite(available on the internet), and Pfam (available on the internet). Eachdescription in The Table begins with the pfam and Prosite identifyingnumbers, the full name of the domain, and a detailed description,including biological and in vivo implications/functions for the domain,references which further describe such implications/functions, andreferences that describe tests/assays to measure theimplications/functions.

10. Single Gene Functions & Utilities Table

The Single Gene Functions & Utilities Table describes particularutilities/functions of interest for individual genes. The Tableidentifies the cDNA_ID of interest, correlates to that cDNA the relevantphenotype, protein domain and microarray/differential expression data.The final column of the Table identifies the utilities/functions ofparticular interest for the identified cDNA.

11. Cluster Functions & Utilities Table

The Cluster Functions & Utilities Table describes particularutilities/functions of interest for identified clusters of genes. TheTable provides the following information:

Record #—an internal identifier.

Group—identifies the group of clusters of interest, wherein each groupis identified with the same utilities/funcions as set forth in theright-hand most column.

cDNA—identifies the cDNA of interest with the noted utility/function.

cDNA_Cluster—identifies the cDNA Cluster ID of interest.

Gi No—refers to the public genomic sequence that matches to the cDNA

NR Hit—refers to the most relevant protein domain for the cDNA ofinterest.

Pfam and Pfam Desc—provide the protein domain name.

Notes/Annotations—provides some notes relevant to the data/informationanalysis.

Utilities/Functions—this rightmost column identifies utilities/functionsof particular interest for the group of cDNAs and clusters.

12. cDNA_Clusters Table

The cDNA_Clusters Table correlates the Ceres cDNA_ID nos. (in numericalorder) with the relevant cDNA cluster which contains each cDNA_ID.

13. Stanford_old_new_cDNA_map Table

During the course of the experiments reported herein, some of the cDNAsequences were assigned new Ceres internal cDNA_id numbers. The cDNA_mapTable provides a list of the original “old” cDNA_ids and correlatesthose id numbers with any new cDNA_id which may have been assigned.Thus, any “old” and “new” cDNA ids which are on the same line in theTable are, in fact, the same sequence.

14. gb_Only_Peptides Table

In the Protein Group table, a number of proteins encoded by Genbankpredictions are included. These proteins were referenced with a peptideID number. The peptide ID number is linked to the amino acid sequence ofthe Genbank prediction in this table.

15. Stanford_Old_New_cDNA Table

During the course of the experiments reported herein, some of the cDNAsequences utilized in the Stanford Microarray differential expressionanalysis experiments were assigned new Ceres internal cDNA_id numbers.The Stanford_old_new_cDNA Table provides a list of the original “old”cDNA_ids and correlates those id numbers with any new cDNA_id which mayhave been assigned. Thus, any “old” and “new” cDNA ids which are on thesame line in the Table are, in fact, the same sequence.

16. Enhanced_Amino Table

This table lists the peptide IDs of polypeptides with enhanced aminoacid content. The table list the peptide ID following with the singleletter code of the amino acid that is enhanced. The table also includesa frequency that the amino acid occurred. The frequency was calculatedby dividing the total number of the desired amino acid indicated in thecolumn by the number of residues in the peptide. For example, if aminoacid A, occurred 50 times in a polypeptide that is 100 amino acid long,the frequency would be 50 divided by 100 or 0.5.

17. Stanford_old_new_cDNA_map Table

During the course of the experiments reported herein, some of the cDNAsequences were assigned new Ceres internal cDNA_id numbers. Thedocket_(—)80090_(—)101_cDNA_map provides a list of the original “old”cDNA_ids in the Reference and Sequence tables and correlates those idnumbers with any new cDNA_id which may have been assigned and utilizedin the remaining tables. Thus, any “old” and “new” cDNA ids which are onthe same line in the Table are, in fact, the same sequence.

II. How the Inventions Reveal how Genes, Gene Components and ProductsFunction

The different experimental molecular genetic approaches focused ondifferent aspects of genes, gene components, and gene products of theinventions. The variety of the data demonstrates the multiple functionsand characteristics of single genes, gene components, and products. Thedata also explain the pathways and networks in which individual genesand products participate and interact. As a result, the circumstances orconditions are now known when these genes and networks are active. Thesenew understandings of biology are relevant for many plant species. Thefollowing section describes the process by which Applicants analyzed theinventions generated by the Ceres Genomic Engine:

II.A. Experimental Results Reveal Many Facets of a Single Gene

The experimental results are used to dissect the function of individualcomponents and products of the genes. For example, the biochemicalactivity of the encoded protein could be surmised from sequenceanalyses, and promoter specificity could be identified throughtranscriptional analyses. Generally, the data presented herein can beused to functionally annotate either the protein sequence and/or theregulatory sequence that control transcription and translation.

II.A.1. Functions of Coding Sequences Revealed by the Ceres GenomicEngine

II.A.1.a. Sequence Similarity to Proteins of Known Function can be Usedto Associate Biochemical Activities and Molecular Interaction to theProteins of the Invention

The protein sequences of the invention were analyzed to determine ifthey shared any sequence characteristics with proteins of knownactivity. Proteins can be grouped together based on sequence similarity,either localized or throughout the length of the proteins. Typically,such groups of proteins exhibit common biochemical activities orinteract with similar molecules.

II.A.1.a.1. Presence of Amino Acid Motifs Indicates Biological Function

Localized protein sequence similarity, also referred to as amino acidmotifs, have been attributed to enzyme or protein functions. A libraryof motifs, important for function, have been documented in PROSITE, apublic database available on the internet. This library includesdescriptions of the motifs and their functions. The zinc finger motif isone such entry in PROSITE, which reports that the zinc finger domain ofDNA-binding proteins is typically defined by a 25-30 amino acid motifcontaining specific cysteine or histidine residues that are involved inthe tetrahedral coordination of a zinc ion. Any protein comprising asequence similar to the zinc finger amino acid motif will have similarfunctional activity (specific binding of DNA).

Protein sequences of the invention have been compared to a library ofamino acid motifs in the pFAM database, which is linked to the PROSITEdatabase. If any of Applicants' protein sequences exhibit similarity tothese amino acid motifs or domains, the Reference Table notes the nameand location of the motif in the “Pred. PP Nom. & Annot” section of theReference tables. A description of any biochemical activities that areassociated to these domains, and therefore associated with Applicants'proteins, is included in the Protein Domain table.

For example, polypeptide, CERES Sequence ID NO: 1545823 is associatedwith zinc finger motif as follows in the Reference Table:

(C) Pred. PP Nom. & Annot.

-   -   Zinc finger, C3HC4 type (RING finger)    -   Loc. Sequence ID NO 133059: 58->106 aa.

II.A.1.a.2. Related Amino Acid Sequences Share Similar BiologicalFunctions

It is apparent, when studying protein sequence families, that someregions have been better conserved than others during evolution. Theseregions are generally important for the function of a protein and/or forthe maintenance of its three-dimensional structure.

The Reference Table reports in section “(Dp) Rel. AA Sequence” when aprotein shares amino acid similarity with a protein of known activity.The section reports the gi number of the protein of known activity, abrief description of the activity, and the location where it sharessequence similarity to Applicants' polypeptide sequence.

Using this analysis, biochemical activity of the known protein isassociated with Applicants' proteins. An example for the polypeptidedescribed above is as follows:

(Dp) Rel. AA Sequence

-   -   Align. NO 524716    -   gi No 2502079    -   Desp.: (AF022391) immediate early protein; ICP0 [Feline        herpesvirus 1]    -   % Idnt.: 33.7    -   Align. Len.: 87    -   Loc. Sequence ID NO 133059: 52->137 aa.

II.A.1.b. Differential Expression Results Explain in which CellularResponses the Proteins of the Invention are Involved

Differential expression results show when the coding sequence istranscribed, and therefore when the activity of the protein is deployedby the cell. Similar coding sequences can have very differentphysiological consequences because the sequences are expressed atdifferent times or places, rather than because of any differences inprotein activity. Therefore, modified levels (increased or decreased) ofexpression as compared to a control provide an indication of thefunction of a corresponding gene, gene components, and gene products.

These experiments can determine which are genes “over-expressed” under agiven stimulus. Such over-expressed genes give rise to higher transcriptlevels in a plant or cell that is stimulated as compared to thetranscript levels of the same genes in a control organism or cell.Similarly, differential expression experiments can reveal“under-expressed” genes.

To increase the cellular response to a stimulus, additional copies ofthe coding sequences of a gene that is over-expressed are inserted intoa cell. Increasing transcript levels of an over-expressed gene caneither heighten or prolong the particular cellular response. A similarenhancement can occur when transcription of an under-expressed gene isinhibited. In contrast, the cellular response will be shortened or lesssevere when the over-expressed genes are inhibited or when expression ofthe under-expressed genes are increased.

In addition to analyzing the levels of transcription, the data were alsoanalyzed to gain insight into the changes in transcription over time.That is, while the plants in the experiments were reacting to either anexternal or internal stimulus, a differential experiment takes asnapshot of the transcription levels in the cells at one specific time.However, a number of snap-shots can be taken at different time pointsduring an external stimulus regime, or at different stages ofdevelopment during an internal stimulus. These results show how theplant changes transcription levels over time, and therefore proteinlevels in response to specific stimuli to produce phenotypic changes.These results show that a protein can be implicated in a single, butmore likely, in a number of cellular responses.

II.A.1.b.I. The Transcript Levels of a Protein Over Time in Response toa Stimuli are Revealed by Transcriptional Analyses Over Many Experiments

Applicants produced data from plants at different times after a specificstimulus. These results show whether the expression level of a genespikes at a key moment during the cellular response, or whether thetranscript level remains constant. Thus, coding sequences not only canbe determined to be over- or under-expressed, but also can be classifiedby the initial timing and duration of differential expression. Thisunderstanding of timing can be used to increase or decrease any desiredcellular response.

Generally, Applicants have assayed plants at 2 to 4 different timepoints after exposing the plants to the desired stimuli. From theseexperiments, “early” and “late” responders were identified. These labelsare applied to either the regulatory sequences driving transcription ofthe gene as well as to the protein encoded by the gene.

The example in FIG. 5 illustrates how the genes, gene components andproducts were classified as either early or late responders following aspecific treatment. The mRNAs from plants exposed to drought conditionswere isolated 1 hour and 6 hours after exposure to drought conditions.These mRNAs were tested utilizing microarray techniques. The graph IFIG. 5 illuminates possible transcription profiles over the time course,plotting all the (+) data points as +1 and all the (−) data points as −1(the value for each time point was determined using a pair of microarraychips as described above).

Data acquired from this type of time course experiment are useful tounderstand how one may increase or decrease the speed of the cellularresponse. Inserting into a cell extra copies of the coding sequence ofearly responders in order to over-express the specific gene can triggera faster cellular response. Alternatively, coding sequences of lateresponders that are over-expressed can be placed under the control ofpromoters of early responders as another means to increase the cellularresponse.

Inserting anti-sense or sense mRNA suppression constructs of the earlyresponders that are over-expressed can retard action of the lateresponders, thereby delaying the desired cellular response. In anotherembodiment, extra copies of the promoters of both early and lateresponders can be added to inhibit expression of both types ofover-expressed genes.

The experiments described herein can be grouped together to determinethe time course of the transcript levels of different coding sequencesin response to different stimuli. Examples of different groups are asfollows (the examples include the IDs for both corn and Arabidopsisexperiments):

-   -   NAA (EXPT IDs 108564, 108565, 108516, 108554)    -   BA (EXPT IDs 108566, 108567, 108517)    -   GA (EXPT IDs 108562, 108563, 108519, 108520, 108521, 108484,        108485, 108486)    -   BR (EXPT IDs 108580, 108581, 108557, 108478, 108479, 108480,        108481)    -   ABA (EXPT IDs 108560, 108561, 108513, 108597)    -   Drought (EXPT IDs 108572, 108573, 108502, 108503, 108504,        108556, 108482, 108483, 108473, 108474, 108477)    -   Cold (EXPT IDs 108578, 108579, 108533, 108534)    -   Heat (EXPT IDs 108576, 108577, 108522, 108523)    -   Osmotic stress (EXPT IDs 108570, 108571, 108541, 108542, 108553,        108539, 108540)    -   Reactive Oxygen (EXPT IDs 108582, 108583, 108537, 108538,        108558)    -   NO (EXPT IDs 108584, 108585, 108526, 108527, 108559)    -   Wounding (EXPT IDs 108574, 108575, 108524, 108525)    -   SA (EXPT IDs 108586, 108587, 108515, 108552, 108471, 108472,        108469, 108470, 107953, 107960, 108443, 108440, 108441, 108475,        108476)    -   MeJA (EXPT IDs 108568, 108569)    -   Finale (EXPT IDs 108467, 107871, 107876)    -   Trimec (EXPT IDs 108466, 107886, 107891)    -   Round-up (EXPT IDs 108465, 107896)    -   Glean (EXPT IDs 108468, 107881)

II.A.1.b.2. The Transcript Levels of a Protein Over DifferentDevelopmental Stages can be Identified by Transcriptional Analyses OverMany Experiments

Differential expression data were produced for different developmentstages of various organs and tissues. Measurement of transcript levelscan divulge whether specific genes give rise to spikes of transcriptionat specific times during development, or whether transcription levelsremain constant. This understanding can be used to increase speed ofdevelopment, or to arrest development at a specific stage.

Like the time-course experiments, the developmental stage data canclassify genes as being transcribed at early or late stages ofdevelopment. Generally, Applicants assayed different organs or tissuesat 2-4 different stages.

Inhibiting under-expressed genes at either early or late stages cantrigger faster development times. The overall development time also canbe increased by this means to allow organs and tissue to grow to alarger size or to allow more organs or tissues to be produced.Alternatively, coding sequences of late stage genes that areunder-expressed can be placed under the control of promoters of earlystage genes to increase heighten development.

Inserting extra copies of the coding sequence early stage genes that areunder-expressed can retard action of the late-stage genes and delay thedesired development.

Fruit development of Arabidopsis is one example that can be studied.Siliques of varying sizes, which are representative of different stages,were assayed by microarray techniques. Specifically, mRNA was isolatedfrom siliques between 0-5 mm, between 5-10 mm and >10 mm in length. Thegraph of FIG. 6 shows expression pattern of a cell wall synthesis gene,cDNAID 1595707, during fruit development.

The developmental course shows that the gene encoding a cell wallsynthesis protein is up-regulated when the fruit is 0-5 mm but returnsto normal levels at 5-10 mm and >10 mm. Increase of cell wall synthesiscan lead to larger cells and/or greater number of cells. This type ofincrease can boost fruit yield. The coding sequence of the cell wallsynthesis protein under the control of a strong early stage promoterwould increase fruit size or number.

A pectinesterase gene was also differentially expressed during fruitdevelopment, cDNA ID 1396123. Pectinesterase catalyzes the hydrolysis ofpectin into pectate and methanol. This biochemical activity plays animportant role in cell wall metabolism during fruit ripening. To shortenthe time for fruit ripening, extra copies of this gene with itsendogenous promoter can be inserted into a desired plant. With itsnative promoter, the extra copies of the gene will be expressed at thenormal time, to promote extra pectinesterase at the optimal stage offruit development thereby shortening ripening time.

A number of Applicant's experiments can be grouped together to studychanges of transcript levels over a number development stages. Below areexamples of groups of experiments:

-   -   a Root, Root Tip, and rhl mutant (EXPT IDs 108594, 108433,        108599, 108434, 108439)    -   Flowers Drought Exposed Flowers, SA Treated Flowers (EXPT IDs        108473, 108474, 108429, 108430, 108431, 108475, 108476, 108501)    -   BR Shoot Apices, Leaves, Stm (EXPT IDs 108478, 108479, 108480,        108481, 108598, 108535, 108536, 108435)    -   Leaf and Stm (EXPT IDs 108477, 108512, 108497, 108498, 108598,        108478, 108479, 108480, 108481, 108598, 108535, 108536, 108435)    -   Imbibded & Germinating Seeds 1, 2, 3, And 4 Days (EXPT IDs        108461, 108462, 108463, 108464, 108528, 108529, 108530, 108531,        108545, 108546, 108547, 108518, 108529, 108543, 108544)    -   Tissue Specific Expression (3 week rosette leaves, Tissue        Specific Expression (3 week stems), Tissue Specific Expression        (2 week roots) (EXPT IDs 108497, 108498, 108439)    -   Tissue Specific Expression (3 week rosette leaves), Germinating        Seeds (EXPT IDs 108497, 108461)    -   Tissue Specific Expression (3 week rosette leaves, stm mutants,        BR_Shoot Apices Expt, root tips, Tissue Specific Expression (2        week roots) (EXPT IDs 108497, 108435, 108480, 108434, 108439)    -   BR_Shoot Apices Expt, root tips, Tissue Specific Expression        (flower buds) (EXPT IDs 108480, 108434, 108431)    -   Arab_Ler-pi_ovule_(—)1, ap2-floral buds, Tissue Specific        Expression (flower buds), Tissue Specific Expression (<5 mm        siliques) (EXPT IDs 108595, 108501, 108431, 108437)    -   Tissue Specific Expression (2 week roots), rhl mutant2, BR_Shoot        Apices Expt, Trichome Inflorescences (EXPT IDs 108439, 108433,        108480, 108452)

II.A.1.b.3. Proteins that are Common in a Number of Similar Responsescan be Identified by Transcriptional Analyses Over a Number ofExperiments

The differential expression experiments also reveal the genes, andtherefore the coding sequence, that are common to a number of cellularresponses. By identifying the genes that are differentially expressed ina number of similar responses, the genes at the nexus of a range ofresponses are discovered. For example, genes that are differentiallyexpressed in all the stress responses are at the hub of many of thestress response pathways.

These types of nexus genes, proteins, and pathways are differentiallyexpressed in many or majority of the responses or developmentalconditions of interest. Typically, a nexus gene, protein, or pathway isdifferentially expressed in generally the same direction in many ormajority of all the desired experiments. By doing so, the nexus gene canbe responsible for triggering the same or similar set of pathways ornetworks for various cellular responses. This type of gene is useful inmodulating pleiotropic effects or triggering or inhibiting a generalclass of responses.

When nexus genes are differentially expressed in a set of responses, butin different directions, these data indicate that a nexus gene isresponsible for creating the specificity in a response by triggering thesame pathway but to a different degree. Placing such nexus genes under aconstitutive promoter to express the proteins at a more constant levelcan remove the fluctuations. For example, a plant that is better droughtadapted, but not cold adapted can be modified to be tolerant to bothconditions by placing under the control of a constitutive promoter anexus gene that is up-regulated in drought but down regulated in cold.

Applicants' experiments can be grouped together to identify such nexusgenes. Examples of these groups are as follows:

-   -   Herbicide Response        -   Trimec, Finale, Glean, Round-up (EXPT IDs 108467, 107871,            107876, 108468, 107881, 108465, 107896, 108466, 107886,            107891)    -   Stress Response        -   Drought, Cold, Heat, Osmotic Stress (EXPT IDs 108578,            108579, 108533, 108534, 108572, 108573, 108502, 108503,            108504, 108556, 108482, 108483, 108473, 108474, 108477,            108576, 108577, 108522, 108523, 108570, 108571, 108541,            108542, 108553, 108539, 108540)        -   Drought, Cold, Heat, PEG, Trimec, Finale, Glean, Round-up            (EXPT IDs 108578, 108579, 108533, 108534, 108572, 108573,            108502, 108503, 108504, 108556, 108482, 108483, 108473,            108474, 108477, 108576, 108577, 108522, 108523, 108570,            108571, 108541, 108542, 108553, 108539, 108540)        -   Wounding, SA, MeJA, Reactive Oxygen, NO (EXPT IDs 108568,            108569, 108555, 108584, 108585, 108526, 108527, 108559,            108582, 108583, 108537, 108538, 108558, 108586, 108587,            108515, 108552, 108471, 108472, 108469, 108470, 107953,            107960, 108443, 108440, 108441, 108475, 108476, 108574,            108575, 108524, 108525)    -   Hormone Responses        -   NAA, BA, BR, GA, TRIMEC (EXPT IDs 108566, 108567, 108517,            108580, 108581, 108557, 108478, 108479, 108480, 108481,            108562, 108563, 108519, 108520, 108521, 108484, 108485,            108486, 108564, 108565, 108516, 108554, 108466, 107886,            107891)        -   NAA, Trimec (EXPT IDs 108566, 108567, 108517, 108580,            108581, 108557, 108478, 108479, 108480, 108481, 108562,            108563, 108519, 108520, 108521, 108484, 108485, 108486,            108564, 108565, 108516, 108554, 108466, 107886, 107891)

II.A.1.b.4. Proteins that are Common to Disparate Responses can beIdentified by Transcriptional Analyses Over a Number of Experiments

Phenotypes and traits result from complex interactions between cellularpathways and networks. Which pathways are linked by expression of commongenes to specify particular traits can be discerned by identifying thegenes that show differential expression of seemingly disparate responsesor developmental stages. For example, hormone fluxes in a plant candirect cell patterning and organ development. Genes that aredifferentially expressed both in the hormone experiments and organdevelopment experiments would be of particular interest to control plantdevelopment.

Examples of Such Pathway Interactions Include:

-   -   (i) The Interaction Between Stress Tolerance Pathways And        Metabolism Pathways;    -   (ii) Interaction Between Hormone Responses And Developmental        Changes In The Plant;    -   (iii) Interactions Between Nutrient Uptake And Developmental        Changes;    -   (iv) Mediation Of Stress Response By Hormone Responses; And    -   (v) Interactions Between Stress Response And Development.        Applicant's experiments can be grouped together to identify        proteins that participate in interacting pathways or networks.        Specific groups of experiments include, for example:

(i) Stress & Metabolism

-   -   Germinating Seeds (Day 1), Arab_(—)0.1uM_Epi-Brass_(—)1,        Arab_NO3_H-to-L_(—)1, Arab_(—)100uM_GA3_(—)1 (EXPT IDs 108461,        108580, 108592, 108562)

(ii) Hormones & Development

-   -   NAA, BA & Root Tips (EXPT IDs 108566, 108567, 108517, 108564,        108565, 108516, 108554, 108434, 108466, 107886, 107891)    -   NAA, Roots & Root Tips (EXPT IDs 108564, 108565, 108516, 108554,        108599, 108434, 108439, 108466, 107886, 107891)    -   NAA, BA, Roots And/Or Root Tips (EXPT IDs 108564, 108565,        108516, 108554, 108599, 108434, 108439, 108466, 107886, 107891,        108566, 108567, 108517)    -   NAA, BA And Leaf (EXPT IDs 108566, 108567, 108517, 108518,        108529, 108512, 108497, 108498, 108598, 108564, 108565, 108516,        108554, 108466, 107886, 107891)    -   NAA, BA, Leaves, Roots And/Or Root Tips (EXPT IDs 108566,        108567, 108517, 108518, 108529, 108512, 108497, 108498, 108598,        108564, 108565, 108516, 108554, 108466, 107886, 107891, 108599,        108434, 108439)    -   ABA & Siliques (Of Any Size) (EXPT IDs 108560, 108561, 108513,        108597, 108436, 108437, 108438)    -   GA, Imbibed & Germinating Seeds, ABA & Siliques (Of Any Size)        (EXPT IDs 108560, 108561, 108513, 108597, 108562, 108563,        108519, 108520, 108521, 108484, 108485, 108486, 108461, 108462,        108463, 108464, 108528, 108529, 108530, 108531, 108545, 108546,        108547, 108518, 108529, 108543, 108544, 108436, 108437, 108438)    -   Tissue Specific Expression (3 week rosette leaves),        Arab_(—)0.1uM_Epi-Brass_(—)1, Arab_(—)100uM_GA3_(—)1,        Germinating Seeds (Day 1), (EXPT IDs 108461, 108497, 108580,        108562, 108461)

(iii) Nutrient Uptake And Development

-   -   Any Or All Nitrogen Experiments With Siliques (Of Any Size)        (EXPT IDs 108592, 108593, 108588, 108589, 108590, 108591,        108532, 108548, 108549, 108550, 108551, 108454, 108455, 108487,        108488, 108489, 108436, 108437, 108438)    -   Any Or All Nitrogen Experiments With Roots Or Root Tips (EXPT        IDs 108518, 108529, 108592, 108593, 108588, 108589, 108590,        108591, 108532, 108548, 108549, 108550, 108551, 108454, 108455,        108487, 108488, 108489, 108594, 108433, 108599, 108434, 108439)

(iv) Stress & Hormones

-   -   ABA, Drought (EXPT IDs 108560, 108561, 108513, 108597, 108572,        108573, 108502, 108503, 108504, 108556, 108482, 108483, 108473,        108474, 108477)    -   ABA, Drought, Cold, Heat, & Wounding (EXPT IDs 108560, 108561,        108513, 108597, 108578, 108579, 108533, 108534, 108572, 108573,        108502, 108503, 108504, 108556, 108482, 108483, 108473, 108474,        108477, 108576, 108577, 108522, 108523, 108574, 108575, 108524,        108525)    -   Tissue Specific Expression (3 week rosette leaves),        Arab_(—)100uM_ABA_(—)1, Ws Arabidopsis Drought 2 days, Ws        Arabidopsis Drought 4 days (EXPT IDs 108497, 108560, 108477,        108482)

(v) Stress & Hormones Stress & Hormones

-   -   Nitrogen High transition to Low, Arab_NO3_H-to-L_(—)1, Tissue        Specific Expression (<5 mm siliques), Tissue Specific Expression        (5-10 mm siliques) (EXPT IDs 108455, 108592, 108437, 108436)

II.A.1.c. Observations of Phenotypic Changes Show What PhysiologicalConsequences Applicants' Proteins can Produce

Another direct means of determining the physiological consequences of aprotein is to make aberrant decreases or increases of its expressionlevel in a cell. To this end, Applicants have produced plants wherespecific genes have been disrupted, or produced plants that include anextra expressed copy of the gene. The plants were then planted undervarious conditions to determine if any visible physiological changes arecaused. These changes then are attributed to the changes in proteinlevels.

II.A.2. Differential Expression Results Explain which External orInternal Stimuli Trigger the Regulatory Sequences

Transcriptional studies can reveal the time and place that genes areexpressed. Typically, regulatory sequences, such as promoters, introns,UTRs, etc., control when and in which cells transcription occurs.Differential studies can explain the temporal- and location-specificregulatory sequences that control transcription.

Using the experiments that are provided herein, one skilled in the artcan choose a promoter or any other regulatory sequence that is capableof facilitating the desired pattern of transcription. For example, if apromoter is needed to give rise to increased levels of transcription inresponse to Auxin, but little expression in response to cytokinin, thenthe promoters of cDNAs that were up-regulated in the Auxin experiments,but down-regulated the cytokinin experiments would be of interest.

Time Course Experiments—Time Sensitive

Evaluation of time-course data as described above is also useful toidentify time-specific promoters. Promoters or regulatory sequences,like the coding sequences, can be classified as early or late respondingaccording to the microarray data. Promoters that facilitate expressionof early or late genes are useful to direct expression of heterologouscoding sequences to modulate the cellular response. In the drought data,promoters from “early” responding genes can be selected to activateexpression of any desired coding sequence. Thus, a coding sequence for asalt-tolerance protein that is not typically expressed early in responseto drought could be linked to an “early” responding promoter to increasesalt tolerance within one hour after exposure to drought conditions.

Developmental Experiments—Time Sensitive

Another class of time-sensitive promoters and other regulatory sequencecan be identified from the experiments examining different developmentalstages. These regulatory sequences can drive transcription ofheterologous sequence at particular times during development. Forexample, expression of stress-responsive genes during fruit developmentcan protect any gain in fruit yield.

Common to Many Pathways—Cause General Effects

Promoters and other regulatory sequence associated with cDNAs that aredifferentially expressed in a number of similar responses can be used tocause general effects. These types of regulatory sequences can be usedto inhibit or increase expression of a desired coding sequence in anumber circumstances. For example, protein that is capable of acting asan insecticide can be placed under the control a general “stress”promoter to increase expression, not only when the plant is wounded, butunder other stress attack.

II.B. Experimental Results Also Reveal Pathways or Networks of Genes

II.B.1. Genes Whose Transcription are Well Coordinated Generally ActTogether to Produce Proteins that Participate in the Same Pathway orNetwork

Patrick Brown, one of the pioneers of microarray chip technology,demonstrated that differential expression experiments can identifygroups of genes that encode proteins that participate the same pathwayor network. The work focused on phosphate accumulation and metabolismgenes in yeast and was published in the paper Ogawa et al., Mol BiolCell (2000) December; 11(12):4309-21. The authors identified bymicroarray analysis 22 genes whose transcription was regulated byphosphate concentration. Promoter analysis of these genes showed that 21of them contained a sequence in their promoters that is recognized by atranscriptional activator that is regulated by phosphate. Further,phenotypic studies were completed by mutational analysis of many ofthese 22 genes in yeast. The mutants were shown to be either severelydeficient in accumulation of inorganic polyphosphate (polyP) and P(i),or associated with normal catabolism of polyP in the yeast vacuole. Thispublication proves that genes with correlated transcriptional profilesdo indeed participate in the same pathway or network.

II.B.1.a. Calculating the Correlation Coefficient Between Pairs of GenesBased on the Differential Expression Data

The differential expression data obtained over many experiments revealthe global pattern of transcription of a gene. Transcription patterns,also referred to as profiles, of two different genes can be compared.From this comparison, a correlation coefficient can be calculated as ameasure of the strength of the relationship between the two profiles.

Transcription profiles can be compared by plotting as a point, thedifferential expression of gene1 on the x-axis and gene 2 on the y-axison one experiment. If all the pairs lie on a regression line therelationship and correlation between the two genes are strong. Thecorrelation coefficient can be calculated using a number of methods. Inthe present case, the Spearman method was utilized.

The correlation coefficient can vary from −1 to 1. The coefficientindicates the strength of the relationship between two mRNA transcriptsof any set of data that is examined. A zero coefficient indicates thatno correlation exists between the transcription profiles of two genes inthe samples examined.

Biologically, a high correlation coefficient indicates that a gene(s)triggers the activation or repression of the correlated genes, or haverelated functional roles. Thus, illumination of the activity of one genecan indicate the activities of the genes with highly correlatedtranscription profiles. This implication is true whether the activity isa biochemical activity, molecular interaction, cellular response, orphysiological consequence.

II.B.1.b. The Complete Linkage Analyses of Differential Identity Geneswith Similar Pattern of Transcription

The complete linkage analysis can build groups (or “clusters”) of geneswhose transcription patterns are highly correlated or co-regulated.

Because genes with related functions are frequently expressed in similarpatterns, utilities or roles can be ascribed for genes (withoutobservation of transformed plants) based on their temporal associationwith other genes of known function (a “guilt-by-association” analysis).Ogawa et al. has used correlated mRNA transcription profiles to identifythe function of proteins of unknown function.

The complete linkage analysis utilizes the correlation coefficients thatare calculated for each pair of genes tested in the microarrayexperiments. A cluster is first seeded with any arbitrary transcripttested on the chip. The seed transcript, for this illustration, isdesignated mRNA#0. Next, a minimum threshold is chosen for allacceptable correlation coefficients. In this case, the threshold usedwas 0.75. A list of potential cluster members is compiled by choosingmRNA transcripts that have a correlation coefficient with mRNA#0 that isgreater than the threshold. No limit is placed on the number of mRNAsthat can be added to a cluster so long as the correlation coefficientmeets the threshold limit criterion.

For this example, assume that four mRNAs were added to the cluster,mRNA_(—)1 to mRNA_(—)4. Once the potential cluster members areidentified, the cDNA IDs of each member is added to the potential listin order its correlation coefficient to mRNA#1, the largest correlationcoefficient first. For this example, let's suppose four mRNAs 1-4 arepotential members, they would be ordered as follows:

Correlation Coefficient MRNA# with mRNA#0 MRNA#1 0.9 MRNA#2 0.8 MRNA#30.78 MRNA#4 0.75

A potential member is accepted into the group, if its correlationcoefficients with all other potential members are all greater than thethreshold. Thus, for mRNA#1 to remain in the group the correlationcoefficient between mRNA#1 and mRNA#2 must be greater than 0.75; andmRNA#1 and #3>0.75; and mRNA#1 and mRNA#4>0.75. Potential clustermembers are removed only after reviewing the correlation coefficients ina specific order where mRNAs are reviewed in the order that they appearon the list.

Consequently, review of the correlation coefficients does not begin withany random pair, such as mRNA#3 and mRNA#4. The review begins betweenmRNA#1 and mRNA#2, which are the top two on the list.

If correlation coefficient between mRNA#1 and mRNA#2 is less than thethreshold, then mRNA#2 is removed from the cluster. mRNA#2 is removedbecause its correlation coefficient with mRNA#0 is 0.8 which is lessthan 0.9, the correlation coefficient of mRNA#1 and mRNA#0.

This illustrates the rule that if the correlation coefficient is lessthan the threshold, then only one of the pair not accepted as a clustermember, specifically, the one with the lower coefficient to the seedmRNA#0.

This process of iterative reviewing of correlation coefficients betweenpotential members continues until all pairs are reviewed. In this case,the coefficient between mRNA#1 and mRNA#3 would be reviewed becausethese are the two highest ones on the list besides mRNA#1 and #2. Thenext pair to be reviewed would be mRNA#1 and #4, etc.

Applicants have analyzed the data using several sets of parameters forthe complete linkage analysis as shown in the table below:

Correlation Coefficient Max number of Method Threshold members in acluster Organism CL_METHOD_TYPE = 0.9 MAX_SIZE = 15 Arabidopsis TRUECL_METHOD_TYPE = 0.75 MAX_SIZE = 30000 Arabidopsis TRUE CL_METHOD_TYPE =0.70 MAX_SIZE = 30000 Arabidopsis TRUE CL_METHOD_TYPE = 0.9 MAX_SIZE =15 Zea TRUE CL_METHOD_TYPE = 0.75 MAX_SIZE = 30000 Zea TRUECL_METHOD_TYPE = 0.70 MAX_SIZE = 30000 Zea TRUE CL_METHOD_TYPE = 0.9MAX_SIZE = 15 Arabidopsis TRUE CL_METHOD_TYPE = 0.75 MAX_SIZE = 30000Arabidopsis TRUE CL_METHOD_TYPE = 0.70 MAX_SIZE = 30000 Arabidopsis TRUECL_METHOD_TYPE = 0.9 MAX_SIZE = 15 Zea TRUE CL_METHOD_TYPE = 0.75MAX_SIZE = 30000 Zea TRUE CL_METHOD_TYPE = 0.70 MAX_SIZE = 30000 ZeaTRUE

The results of these cluster analyses are reported in the MA_clusttable.

II.B.1.c. The Nearest Neighbor Analyses of Differential Group Genes withCorrelated but Dissimilar Transcription Profiles

The nearest neighbor analysis differs from the complete linkagealgorithm by not requiring all members to meet the correlation thresholdwith each other. Thus, a member of a nearest neighbor cluster need onlybe closely correlated to one other member of the cluster. It is not evenrequired that all members be closely correlated to the seed mRNAtranscript.

In a complete linkage cluster all the transcription profile of allmembers are correlated to a greater or lesser extent. In contrast, acluster deduced by the nearest neighbor analysis may include memberswith differing transcription profiles. However, nearest neighbor bringsto light clusters of interacting genes. In the nearest neighboranalysis, the seed mRNA may not have a very high correlation coefficientwith the last mRNA added to the cluster.

The nearest neighbor analysis, like the complete linkage analysis, isinitiated by seeding each cluster with a mRNA_(—)0. The cluster size isdetermined by setting a threshold coefficient and setting a limit on thenumber of members that can be added to the cluster.

The cluster is expanded in an iterative fashion determining which mRNAhas the highest correlation coefficient with mRNA_(—)0. The additionalmember is labeled mRNA_(—)1. Next, a list of potential candidates isgenerated by finding the mRNA that has the highest correlation tomRNA_(—)0 (besides mRNA_(—)1) and finding the mRNA that has the highestcoefficient with mRNA_(—)1. Whichever of the candidates has the highestcorrelation coefficient is added to the cluster. Then, a list of threepotential candidates is generated similarly.

Addition of members continues until either (1) all the correlationcoefficients of potential members is lower than the threshold or (2)number of members in the cluster meets the size limitation.

Applicants have analyzed the data using several sets of parameters forthe nearest neighbor analysis as shown in the table below:

Correlation Max number of Coefficient members in a Method Thresholdcluster Organism NN_METHOD_TYPE = TRUE 0.5 MAX_HITS = 15 ArabidopsisFULL_NN_METHOD_TYPE = 0.8 NONE Arabidopsis TRUE FULL_NN_METHOD_TYPE =0.6 NONE Arabidopsis TRUE NN_METHOD_TYPE = TRUE 0.5 MAX_HITS = 15 ZeaFULL_NN_METHOD_TYPE = 0.8 NONE Zea TRUE FULL_NN_METHOD_TYPE = 0.6 NONEZea TRUE NN_METHOD_TYPE = TRUE 0.5 MAX_HITS = 15 ArabidopsisFULL_NN_METHOD_TYPE = 0.8 NONE Arabidopsis TRUE FULL_NN_METHOD_TYPE =0.6 NONE Arabidopsis TRUE NN_METHOD_TYPE = TRUE 0.5 MAX_HITS = 15 ZeaFULL_NN_METHOD_TYPE = 0.8 NONE Zea TRUE FULL_NN_METHOD_TYPE = 0.6 NONEZea TRUE

The results of these cluster analyses are reported in the MA_clusttable.

II.C. Experimental Results Reveal the Functions and Characteristics ofGenes, Pathways and Networks

II.C.1. Linking Biochemical or Metabolic Activities of One Protein in aCluster to the Other Proteins in the Same Microarray Cluster

As shown in the Ogawa et al., Mol Biol Cell (2000), genes whosetranscription profiles cluster together as being strongly correlatedtypically take part in the same pathway or network. Thus, the activityof one gene in the cluster can be associated to the other genes in thecluster with highly correlated transcription profiles. This associationis true whether the activity is a biochemical activity, molecularinteraction, cellular response or physiological consequence.

One example of this is cluster 420 of the report (shown below). In thiscluster, a protein encoded by cDNA ID 1025791 did not match to any pFAMdomain. However, through the microarray data, the gene that encodes thatprotein had a transcription profile that was correlated with other genesthat encode ribosomal proteins. Thus, the activity of the ribosomalgenes can be associated with the protein with no pFAM match. All theproteins in the same cluster would be associated with mRNA translationand protein synthesis.

II.C.2 Using Differential Expression Data to Determine when the Genesand Pathways are Active

The differential expression data can be used to associate the cellularresponse that results when the clusters of genes are transcribed. Forthe complete linkage clusters, the genes in the cluster will producesimilar transcription profiles. The experiments where the genes in thecluster are differentially expressed as compared to the control definethe cellular responses that all the genes of the cluster are capable ofmodulating.

For example, for the cluster shown above, the mRNA levels for the geneswere significantly different in the nitrogen response experiments. Thus,the data shows that this cluster of genes is associated with proteinsynthesis in response to nutrient uptake.

II.C.3. Using Phenotype Data to Determine when Genes and Pathways areActive

The phenotypic data can be used to demonstrate the physiologicalconsequences of that result when a cluster of genes is active. Whetherthe clusters were generated by the complete linkage or the nearestneighbor analyses, if a single gene in the cluster has been implicatedin phenotypic changes, then any one or combination of the other genes inthe cluster can also modulate the same or similar phenotypic changes.

Utilities of Particular Interest

The following sections describe utilities/functions for the genes, genecomponents and products of the invention. The sequences of theinvention, as discussed above, can be recognized as a particular type ofgene (e.g. root gene, leaf gene, etc.) by means of particular termsutilized in the Knock-in and Knock-out Tables and by the results of thedifferential expression experiments. Combined analysis of those dataalso identify genes with utilities/functions of particular interest. TheSingle Gene Functions and Utilities Table correlates that data andspecific genes with those utilities/functions of particular interest.

Utilities of Particular Interest for Clustered Sequences

As discussed further herein, the genes, gene components and products ofthe invention have been clustered together into groups. This enables oneto understand the function/utility of one member of the cluster basedupon knowledge about one or more other members of the cluster. Inaddition, this enables an understanding of some utilities/functions of acluster that would be of particular interest. The Cluster Functions andUtilities Table lists some of the clusters of the invention and notesthe functions/utilities that are of particular interest for each of theclusters. Of course, these functions/utilities are of particularinterest for each member of each particular cluster.

II.D. Experimental Results Provide an Understanding of Genes, Pathwaysand Networks in Many Plant Species

By analyzing the constant and variable properties of groups of similarsequences, it is possible to derive a structural and functionalsignature for a protein family, which distinguishes its members from allother proteins. This approach has allowed the Applicants to assignproteins into functional groups and identify orthologous proteins bothwithin and between species. A pertinent analogy to be considered is theuse of fingerprints by the police for identification purposes. Afingerprint is generally sufficient to identify a given individual.Similarly, a protein signature can be used to assign a newly sequencedprotein to a specific family of proteins and thus to formulatehypotheses about its function.

Proteins can be grouped together because they share a single motif ormany motifs. Typically, proteins that share a series of motifs sharegreater functional equivalence. Usually, signature sequences comprisemore than one motif in a particular order from N-terminus to C-terminus.

A list of these groups can be found in the Protein Group Table. Thesequences were grouped together using the iterative protein sequencelocal alignment software, PSI-BLAST. This software begins by aligning anumber sequences where the probability that the alignment occurred bychance is set by a threshold e-value. In the Applicants' case, thethreshold e-value was set at 10⁻⁵⁰, 10⁻³⁰, and 10⁻¹⁰. The algorithmgenerates a consensus sequence from the sequences that were alignedtogether. The consensus sequence was then used to find sequences thatmatched to it with a probability that was less than the set threshold.The algorithm performs the iterative tasks of aligning and generating aconsensus sequence any number of times. Generally, Applicants performedone iteration for the 10⁻¹⁰ e-value threshold, two iterations for the10⁻³⁰ threshold, and three iterations for the 10⁻⁵⁰ threshold.

Each group can contain sequences from one of more organisms. The groupsincluded both Ceres polypeptides and public polypeptide sequences. TheCeres polypeptides are identified by their Ceres Sequence ID NO aslisted in the Reference Table.

Each group contains sequences that were included at the 10⁻⁵⁰, 10⁻³⁰,and 10⁻¹⁰ e-value cutoffs. For each group, the peptide ID and at whichcutoff the peptide was included into the group. The same peptide ID maybe included in the group three times as peptide ID 50, peptide ID 30 andpeptide ID 10. The data indicates that peptide ID was included in thegroup when the threshold was either 10⁻⁵⁰, 10⁻³⁰, or 10⁻¹⁰. All thepeptide IDs that are followed by “50” were included in the protein groupwhen the e-value cutoff was 10⁻⁵⁰. All the peptide IDs that are followedby either “30” or “50” were included in the protein group when thethreshold e-value was 10⁻³⁰. All the peptide IDs that are followed by“10”, “30” or “50” were included in the protein group when 10⁻¹⁰ wasused as the e-value cutoff.

II.D.1. Conserved Sequences Between Proteins of Different Species GiveRise to a Signature Sequence

The signature sequence for each group of proteins, also referred to asthe consensus sequence. The signature sequence comprises the amino acidsthat are conserved throughout all the proteins in a particular proteingroup. The data are shown in the Protein Group table.

Not all the polypeptides in a group are the same length. Thus, somemembers of the group may not contain the entire signature sequence.However, throughout the length of any member protein, its sequence willmatch the signature sequence.

The consensus sequence contains both lower-case and upper-case letters.The upper-case letters represent the standard one-letter amino acidabbreviations. The lower case letters represent classes of amino acids:

-   -   “t” refers to tiny amino acids, which are specifically alanine,        glycine, serine and threonine.    -   “p” refers to polar amino acids, which are specifically,        asparagine and glutamine    -   “n” refers to negatively charged amino acids, which are        specifically, aspartic acid and glutamic acid    -   “+” refers to positively charged residues, which are        specifically, lysine, arginine, and histidine    -   “r” refers to aromatic residues, which are specifically,        phenylalanine, tyrosine, and tryptophan,    -   “a” refers to aliphatic residues, which are specifically,        isoleucine, valine, leucine, and methonine

In addition to each consensus sequence, Applicants have generated ascoring matrix to provide further description of the consensus sequence.The matrix reports the identity and number of occurrences of all theamino acids that were found in the group members for every residueposition of the signature sequence. The matrix also indicates for eachresidue position, how many different organisms were found to have apolypeptide in the group that included a residue at the relevantposition. These results are reported in the Protein Group Matrix table.

Functional equivalents share similar (1) structural characteristics; (2)biochemical activities and molecular interactions; (3) cellularresponses or activities; or (4) phenotypic effects.

II.D.2. Linking Signature Sequences to Conservation of StructuralCharacteristics

Proteins with related functions show similar three-dimensionalstructures but may not show extensive amino acid sequence similarity.Typically, proteins need only share a single motif or low similarity inmultiple domains to exhibit similar structural features, such as alphahelix, beta sheet, charge residues, stretches of hydrophobicity, etc.Conserved structural features have been implicated in ligand binding byreceptor proteins, binding to a class of substrates, polynucleotidebinding, or protein-protein interactions.

Based on the signature sequences and the Matrix Tables described herein,a number of motifs can be discerned. Motifs are identified as regions inthe signature sequence which are constant in a majority of the membersof the group. Example motifs can be found among Applicant's data whichare shared in the range of 75% to 95% of group members

Typically, a region of the consensus sequence is constant if, at eachposition of the region, the preferred amino acid is chosen from a singleclass of amino acids; even more typically, the preferred amino acid is asingle amino acid. The region can contain a number of positions where anamino acid can be chosen. However, these variable positions are usuallyless than 15% of the total number of residues in the region; moreusually, less than 10%; even more usually, less than 5%.

Generally, a domain is considered to be well defined if the consensussequence is constructed from sequences from at least 2 organisms; morepreferably, at least 3 organisms; even more preferably four organisms orgreater.

Primary domains are best identified from the data presented for the10⁻¹⁰ probability criteria. Using this parameter, the largest number ofproteins is associated into a group. Consequently, the signaturesequence exhibits the greatest amount of variability. The conservedregions, the domains or motifs of the signature contrast against thevariable regions. These variable regions become obvious when sequencesfrom more proteins are compared.

Signature sequences revealed in the 10⁻³⁰ and 10⁻⁵⁰ e-value classes showmore conservation in the domains, and can even display a degree ofconservation in what is considered the variable regions in the 10⁻¹⁰analyses. These more extensively-conserved domains can reflect highersimilarity in function—completely orthologous functions. Proteins thatshare a number of conserved domains, in the same relative order from Nterminus to C terminus, are even more likely to be completelyorthologous. Nevertheless, because of the natural divergence that occursin non-conserved regions during evolution and species differentiation,orthologs can be proteins with only the domains conserved and thereforebe present in the 10⁻³⁰ and 10⁻¹⁰ p value classes of the Ortholog Table.

II.D.3. Linking Signature Sequences to Conservation of BiochemicalActivities and Molecular Interactions

Proteins that possess the same defined domains or motifs are likely tocarry out the same biochemical activity or interact with a similar classof target molecule, e.g., DNA, RNA, proteins, etc. Thus, the pFAMdomains listed in the Reference Tables are routinely used as predictorsof these properties. Substrates and products for the specific reactionscan vary from protein to protein. Where the substrates, ligands, orother molecules bound are identical the affinities may differ betweenthe proteins. Typically, the affinities exhibited by differentfunctional equivalents varies no more than 50%; more typically, no morethan 25%; even more typically, no more than 10%; or even less.

Proteins with very similar biochemical activities or molecularinteractions will share similar structural properties, such as substrategrooves, as well as sequence similarity in more than one motif. Usually,the proteins will share at least two motifs of the signature sequence;more usually, three motifs; even more usually four motifs or greater.Typically, the proteins exhibit 70% sequence identity in the sharedmotifs; more typically, 80% sequence identity; even more typically, 90%sequence identity or greater. These proteins also often share sequencesimilarity in the variable regions between the constant motif regions.Further, the shared motifs will be in the same order from amino- tocarboxyl-termini. The length of the variable regions between the motifsin these proteins, generally, is similar. Specifically, the number ofresidues between the shared motifs in these proteins varies by less than25%; more usually, does not vary by less than 20%; even more usually,less than 15%; even more usually less than 10% or even less.

II.D.4. Linking Signature Sequences to Conservation of CellularResponses or Activities

Proteins that exhibit similar cellular response or activities willpossess the structural and conserved domain/motifs as described in theBiochemical Activities and Molecular Interactions above.

Proteins can play a larger role in cellular response than just theirbiochemical activities or molecular interactions suggest. A protein caninitiate gene transcription, which is specific to the drought responseof a cell. Other cellular responses and activities include: stressresponses, hormonal responses, growth and differential of a cell, cellto cell interactions, etc.

The cellular role or activities of protein can be deduced bytranscriptional analyses or phenotypic analyses as well as bydetermining the biochemical activities and molecular interactions of theprotein. For example, transcriptional analyses can indicate thattranscription of gene A is greatly increased during flower development.Such data would implicate protein A encoded by gene A, in the process offlower development. Proteins that shared sequence similarity in morethan one motif would also act as functional equivalents for protein Aduring flower development.

II.D.5. Linking Signature Sequences to Conservation of PhenotypicEffects

Typically, proteins that are grouped together under the most stringentparameters, e-value≦10⁻⁵, are likely orthologs and therefore, whenpresent in the same or equivalent cells can cause similar phenotypicconsequences. These proteins have very high sequence similarity.Typically, if one of the members of a group is an Arabidopsis protein,then the corn ortholog can rescue an Arabidopsis mutant plant that doesnot produce the Arabidopsis protein. The mutant plant would be rescuedas the parental “wild-type” phenotype by expression of a coding sequenceof the corn protein of the same orthologous group when present in theappropriate cell types of the plant.

Preferably, these functional equivalents have sequence motif identitythroughout much of the length of the protein. However, proteins thatshare very high similarity between a number, usually more than two; evenmore usually, more than three motifs can act as functional equivalentsto produce similar phenotypic effects.

A gene can have coding sequence similarity, i.e., is a homologous. Thecoding sequence can be sufficient to act as a functional equivalent,although the gene as a whole is not an ortholog. For example, twosimilar dwf4 coding sequences were found in the Arabidopsis genome.However, this pair of coding sequences had different promoters and hencedifferent roles in Plantae. But when one of the pair was placed underthe control of its mates' promoter, the phenotypic effects were similarto the effects produced by its mate coding sequence. Therefore, thecoding sequence, but not the genes are orthologous.

III. Description of the Genes, Gene Components and Products, Togetherwith their Use and Application

As described herein, the results of Applicant's experiments provide anunderstanding of the function and phenotypic implications of the genes,gene components and products of the present invention. Bioinformaticanalysis provides such information. The sections of the presentapplication containing the bioinformatic analysis, together with theSequence and Reference Tables, teach those skilled in the art how to usethe genes, gene components and products of the present invention toprovide plants with novel characteristics. Similarly, differentialexpression analysis provides additional such information and thesections of the present application on that analysis; together with theMA_Diff Tables and MA_Cluster Tables, describe the functions of thegenes, gene components and products of the present invention which areunderstood from the results of the differential expression experiments.The same is true with respect to the phenotype data, wherein the resultsof the Knock-in and Knock-out experiments and the sections of thepresent application on those experiments provide the skilled artisanwith further description of the functions of the genes, gene componentsand products of the present invention.

As a result, one reading each of these sections of the presentapplication as an independent report will understand the function of thegenes, gene components and products of the present invention. But thosesections and descriptions can also be read in combination, in anintegrated manner, to gain further insight into the functions and usesfor the genes, gene components and products of the present invention.Such an integrated analysis does not require extending beyond theteachings of the present application, but rather combining andintegrating the teachings depending upon the particular purpose of thereader.

Some sections of the present application describe the function of genes,gene components and products of the present invention with reference tothe type of plant tissue (e.g. root genes, leaf genes, etc.), whileother sections describe the function of the genes, gene components andproducts with respect to responses under certain conditions (e.g.Auxin-responsive genes, heat-responsive genes, etc.). Thus, if onedesires to utilize a gene understood from the application to be aparticular tissue-type of gene, then the condition-specificresponsiveness of that gene can be understood from the differentialexpression tables, and very specific characteristics of actions of thatgene in a transformed plant will be understood by recognizing theoverlap or intersection of the gene functions as understood from the twodifferent types of information. Thus, for example, if one desires totransform a plant with a root gene for enhancing root growth andperformance, one can know the useful root genes from the resultsreported in the knock-in and knock-out tables. A review of thedifferential expression data may then show that a specific root gene isalso over-expressed in response to heat and osmotic stress. The functionof that gene is then described in (1) the section of the presentapplication that discusses root genes, (2) the section of the presentapplication that discusses heat-responsive genes, and (3) the section ofthe application that discusses osmotic stress-responsive genes. Thefunction(s) which are commonly described in those three sections willthen be particularly characteristic of a plant transformed with thatgene. This type of integrated analysis of data can be viewed from thefollowing schematic that summarizes, for one particular gene, thefunction of that gene as understood from the phenotype and differentialexpression experiments.

Gene function known Gene function known Gene function known fromphenotype from first differential from second differential experimentsexpression experiment expression experiment Function A Function AFunction A Function B Function C Function C Function D Function EFunction F Function F Function F Function G Function G Function HFunction I Function I Function J

In the above example, one skilled in the art will understand that aplant transformed with this particular gene will particularly exhibitfunctions A and F because those are the functions which are understoodin common from the three different experiments.

Similar analyses can be conducted on various genes of the presentinvention, by which one skilled in the art can effectively modulateplant functions depending upon the particular use or conditionsenvisioned for the plant.

III.A. Organ-Affecting Genes, Gene Components, Products (IncludingDifferentiation and Function)

III.A.1. Root Genes, Gene Components and Products

The economic values of roots arise not only from harvested adventitiousroots or tubers, but also from the ability of roots to funnel nutrientsto support growth of all plants and increase their vegetative material,seeds, fruits, etc. Roots have four main functions. First, they anchorthe plant in the soil. Second, they facilitate and regulate themolecular signals and molecular traffic between the plant, soil, andsoil fauna. Third, the root provides a plant with nutrients gained fromthe soil or growth medium. Fourth, they condition local soil chemicaland physical properties.

III.A.1.a. Identification of Root Genes

Root genes identified herein are defined as genes, gene components andproducts capable of modulating one or more processes in or functions ofthe root as described below. They are active or potentially active to agreater extent in roots than in most other organs of the plant. Thesegenes and gene products can regulate many plant traits from yield tostress tolerance. That single genes usually affect the development andfunction of roots and whole plants is a consequence of biologicalcellular complexity and the role roots play in supporting the growth ofwhole plants. Examples of such root genes and gene products are shown inthe Reference and Sequence Reference and Sequence Tables and sequencesencoding polypeptides of the Protein Group and Protein Group Matrixtables or fragments thereof, the Knock-In and Knock-Out Tables, and theMA-diff Tables. The function of many of the protein products gained fromcomparisons with proteins of known functions, are also given in the REFTables.

Root Genes Identified by Phenotypic Observations

Root genes are active or potentially active to a greater extent in rootsthan in some other organs/tissue of the plant. Some of the root genesherein were discovered and characterized from a much larger set of genesin experiments designed to find genes that cause phenotypic changes inroot morphology. Such morphological changes include primary and lateralroot number, size and length, as well as phenotypic changes of otherparts of that plant associated with changes in root morphology.

In these experiments, root genes were identified by either (1) ectopicexpression of a cDNA in a plant or (2) mutagenesis of the plant genome.The plants were then cultivated under standardized conditions and anyphenotypic differences recorded between the modified plants as comparedwith the parent plant. The gene(s) causing the changes were deduced fromthe cDNA inserted or disrupted gene. Phenotypic differences wereobserved in:

Primary Roots And Root System

-   -   Size, Including Length And Girth    -   Number    -   Branching    -   Root Waving/Curling Characteristics    -   Gravitropism Changes    -   Agravitropic

Lateral Roots

-   -   Size, Including Length And Girth    -   Number    -   Branching

Results from screening for these phenotypic changes are reported in theKnock-in and Knock-out Tables. Therefore, any sequence reported in thoseTables with one of the above-noted observations is considered a “rootgene”. A “root gene” is also a sequence which, in the Ortholog Tables orin the MA-clust Tables, is grouped/clustered together with at least onesequence that is identified as such by means of the Knock-in andKnock-out Tables.

Root Genes Identified by Differential Expression

Root genes were also identified by measuring the relative levels of mRNAproducts in the root versus the aerial portion of a plant. Specifically,mRNA was isolated from roots and root tips of Arabidopsis plants andcompared to mRNA isolated from the aerial portion of the plantsutilizing microarray procedures. The MA_diff Table(s) reports thetranscript levels of the experiment (see EXPT ID: 108594, 108433,108599, 108434, 108439). For transcripts that had higher levels in thesamples than the control, a “+” is shown. A “−” is shown for whentranscript levels were reduced in root tips as compared to the control.For more experimental detail see the Example section below.

Roots genes are those sequences that showed differential expression ascompared to controls, namely those sequences identified in the MA_difftables with a “+” or “−” indication.

Roots Genes Identified by Cluster Analyses of Differential Expression

Roots Genes Identified by Correlation to Genes that are DifferentiallyExpressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of Roots genes is any group in the MA_clust thatcomprises a cDNA ID that also appears in Expt ID 108594, 108433, 108599,108434, 108439 of the MA_diff table(s).

Roots Genes Identified by Correlation to Genes that Cause PhysiologicalConsequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of Rootsgenes. A group in the MA_clust is considered a Roots pathway or networkif the group comprises a cDNA ID that also appears in Knock-in orKnock-out tables that causes one or more of the phenotypes described insection above.

Roots Genes Identified by Amino Acid Sequence Similarity

Roots genes from other plant species typically encode polypeptides thatshare amino acid similarity to the sequences encoded by corn andArabidopsis Roots genes. Groups of Roots genes are identified in theProtein Group table. In this table, any protein group that comprises apeptide ID that corresponds to a cDNA ID member of a Roots pathway ornetwork is a group of proteins that also exhibits Rootsfunctions/utilities.

Examples of phenotypes, biochemical activities, and transcriptionprofiles that can be modulated by these genes and gene products aredescribed above and below.

III.A.1.b. Use of Root Genes to Modulate Phenotypes

The root genes of the instant invention are capable of modulating one ormore processes of root structure and/or function including (1)development; (2) interaction with the soil and soil contents; and (3)transport in the plant.

Root genes and gene products can be used to alter or modulate one ormore of the following phenotypes.

1.) Development

Roots arise from meristem cells that are protected by a root cap duringroot elongation, but as the root grows out, the cap cells abscise andthe remaining cells differentiate to the tip. Depending on the plantspecies, some surface cells of roots can develop into root hairs. Someroots persist for the life of the plant; others gradually shorten as theends slowly die back; some may cease to function due to externalinfluences. The root genes and gene products of this invention areuseful to modulate any one or all of these growth and developmentprocesses generally, as in root density and root growth; including rate,timing, direction and size.

Root genes and gene products are useful to modulate either the growthand development or other processes in one or more of the following typesof roots, including primary, lateral, and adventitious.

Root genes and gene products are useful to modulate cellular changes incell size, cell division, rate direction and/or number, cell elongation,cell differentiation, lignified cell walls, epidermal cells, such astrichoblasts, and root apical meristem cells (growth and initiation).

Parts of roots (i.e. root architecture) can be modulated by these genesroot and gene products to affect root architecture in, for example, theepidermis cortex (including the epidermis, hypodermis, endodermis,casparian strips, suberized secondary walls, parenchyma, andaerenchyma), stele (including vaculature, xylem, phloem, and pericycle),vasculature, xylem, phloem, root cap, root apical meristem, elongatingregion, and symmetry.

The polynucleotides and polypeptides of this invention can be used tocontrol the responses to internal plant and root programs as well as toenvironmental stimuli in the seminal system, nodal system, hormonesystems (including Auxin and cytokinin), root cap abscission, rootsenescence, gravitropism, coordination of root growth and developmentwith that of other organs (including leaves, flowers, seeds, fruits,stems, and changes in soil environment (including water, minerals, ph,and microfauna and flora).

2.) Interaction with Soil and Soil Contents

Roots are sites of intense chemical and biological activities and as aresult can strongly modify the soil they contact. Roots coat themselveswith surfactants and mucilage to facilitate these activities.Specifically, roots are responsible for nutrient uptake by mobilizingand assimilating water, organic and inorganic compounds, ions andattracting and interacting with beneficial microfauna and flora. Rootsalso help to mitigate the effects of toxic chemicals, pathogens andstress. Examples of root properties and activities that the genes andgene products of this invention are useful to modulate are rootsurfactants and mucilage (including mucilage composition, secretion rateand time, surfactant); nutrient uptake of water, nitrate and othersources of nitrogen, phosphate, potassium, and micronutrients (e.g.iron, copper, etc.); microbes and nematodes associations (such asbacteria including nitrogen-fixing bacteria, mycorrhizae, andnodule-forming and other nematodes); oxygen (including transpiration);detoxification of iron, aluminum, cadium, mercury, salt, and other heavymetals and toxins); pathogen interactions/control (including chemicalrepellents (includes glucosinolates (GSL), which releasepathogen-controlling isothiocyanates); and changes in soil properties,(such as Ph, mineral depletion, and rhizosheath).

3) Transport of Materials in Plants

Uptake of nutrients by roots produces a “source-sink” effect in a plant.The greater the source of nutrients, the larger “sinks,” such as stems,leaves, flowers, seeds, fruits, etc. can grow. Thus, root genes and geneproducts are useful to modulate the vigor and yield of the plant overallas well as distinct cells, organs, or tissues. The root genes and geneproducts are, therefore, useful to modulate vigor (including plantnutrition, growth rate (such as whole plant, including height, floweringtime, etc.), seedling, coleoptile elongation, young leaves, stems,flowers, seeds, fruit, and yield (including biomass (such as fresh anddry weight during any time in plant life, including maturation andsenescence), root/tuber yield (such as number, size, weight, harvestindex, content and composition, (i.e. amino acid, jasmonate, oil,protein and starch), number of flowers, seed yield, number, size,weight, harvest index, content and composition (e.g. amino acid,jasmonate, oil, protein and starch), and fruit yield (such as number,size, weight, harvest index, post harvest quality, content andcomposition, (e.g. amino acid, jasmonate, oil, protein and starch).

Additional Uses of Plants with Modified Roots

Plants with roots modified in one or more of the properties describedabove are used to provide:

-   -   A. Higher vigor and yield of plants and harvested products due        to pathogen resistance from conditioning the soil with        plant-derived chemicals and/or more tolerance to stresses such        as drought, flooding and anoxia.    -   B. Better Animal (Including Human) Nutrition    -   C. Improved Dietary Mineral Nutrition    -   D. Better Plant Survival        -   (a) Decreased Lodging        -   (b) More Efficient Transport        -   (c) More Efficient Physiology        -   (d) More Efficient Metabolism    -   E. Better Resistance To Plant Density Effects    -   F. Increased Yield Of Valuable Molecules    -   G. More Efficient Root Nodulation    -   H. Better Access To Rhizobia Spray Application, For Anaerobic        Soils    -   I. Easier Crop Harvesting And Ground Tillage    -   J. Decreased Soil Erosion

To regulate any of the phenotype(s) above, activities of one or more ofthe root genes or gene products is modulated and tested by screening forthe desired trait. Specifically, the gene, mRNA levels, or proteinlevels can be altered in a plant utilizing the procedures describedherein and the phenotypes can be assayed. As an example, a plant can betransformed according to Bechtold and Pelletier (1998, Methods. Mol.Biol. 82:259-266) and/or screened for variants as in Winkler et al.(1998) Plant Physiol 118: 743-50 and visually inspected for the desiredphenotype or metabolically and/or functionally assayed according toDolan et al. (1993, Development 119: 71-84), Dolan et al. (1997,Development 124: 1789-98), Crawford and Glass (1998, Trends PlantScience 3: 389-95), Wang et al. (1998, PNAS USA 95: 15134-39), Gaxiolaet al. (1998, PNAS USA 95: 4046-50), Apse et al. (1999, Science 285:1256-58), Fisher and Long (1992, Nature 357: 655-60), Schneider et al.(1998, Genes Devel 12: 2013-21) and Hirsch (1999, Curr Opin Plant Biol.2: 320-326).

III.A.1.c. Use of Root Genes to Modulate Biochemical Activities

The activities of one or more of the root genes can be modulated tochange biochemical or metabolic activities and/or pathways such as thosenoted below. Such biological activities can be measured according to thecitations included in the Table below:

BIOCHEMICAL OR METABOLIC ACTIVITIES CITATIONS INCLUDING PROCESS AND/ORPATHWAYS ASSAYS Association Of Root Cell-Cell Recognition Gage et al.(1996) J Bacteriol Morphology With Nitrogen Cell Wall Degradation 178:7159-66 Fixing Bacteria Primary Root, Lateral Cell Division/ElongationSchneider et al. (1998) Genes Root, And Root Hair Cell DifferentiationDevel 12: 2013-21 Initiation Cell Expansion Casimiro et al. (2001).Plant Spacing Auxin Mediated Response Cell 13: 843-852. ElongationPathways Rogg et al. (2001). Plant Branching Cell 13: 465-480. Gaedekeet al. (2001). EMBO J. 20: 1875-1887. Neuteboom et al. (1999). PlantMol. Biol. 39: 273-287. Schindelman et al. (2001). Genes and Dev. 15:1115-1127. Rashotte et al. (2001) Plant Cell 13: 1683-1697. Zhang et al.(2000). J Exp Bot 51: 51-59. Zhang et al. (1998) Science 279: 407-409.Metabolism Organic Molecule Export Moody et al. (1988) Phytochemistry27: 2857-61. Ion Export Uozumi et al. (2000) Plant Physiol 122: 1249-59Frachisse et al. (2000) Plant J 21: 361-71 Nutrient Uptake Frachisse etal. (2000) Plant J 21: 361-71 Uozumio et al. (2000) Plant Physiol 122:1249-59 Williamson et al. (2001). Plant Physiol. 126: 875-882. Zhang etal. (2000). J Exp Bot 51: 51-59. Zhang et al. (1998). Science 279:407-409. Coruzzi et al. (2001). Plant Physiol. 125: 61-64. RootGravitropism And Reactive Oxygen Species Joo et al. (2001) Plant Waving(ROS) Such As Superoxide Physiol. 126: 1055-60. Anions And H2O2 Vitha etal. (2000). Plant Production Physiol. 122: 453-461. Auxin TransportPathways Tasaka et al. (2001) Int Rev Flavonoid Inhibition Of Cytol 206:135-54. Auxin Transport Function Brown et al. (2001) Plant Changes InRoot Cap Ph Physiol 126: 524-35. Starch Synthesis And Fasano et al.(2001) Plant Storage Cell 13: 907-22. Cell Differentiation MacCleery etal. (1999). Cell Elongation Plant Physiol 120: 183-92 Blancaflor et al.(1998). Plant Physiol 116: 213-22 Schneider et al. (1998) Genes Devel12: 2013-21

Other biological activities that can be modulated by the root genes andgene products are listed in the Reference tables. Assays for detectingsuch biological activities are described in the Protein Domain table.

III.A.1.d. Use of Root Genes to Modulate Transcription Levels of PlantGenes

Many genes are “up regulated” or “down regulated” because they belong tonetworks or cascades of genes. Thus some root genes are capable ofregulating many other gene activities via these networks and hencecomplex phenotypes. Examples of transcription profiles of root genes aredescribed in the Table below with associated biological activities.“Up-regulated” profiles are those where the concentrations of the mRNAin total mRNA are higher in roots as compared to aerial parts of aplant; and vice-versa for “down-regulated” profiles.

EXAMPLES OF TRANSCRIPT PHYSIOLOGICAL BIOCHEMICAL LEVELS TYPE OF GENESCONSEQUENCES ACTIVITY Up Regulated Genes Expressed In Primary Root,Transporters Transcripts Root Development Lateral Root, and/or MetabolicEnzymes Responders To Root Hair Growth Change In Cell Micro-Organismaland Differentiation Membrane Structure Symbionts And Microorganism AndPotential Parasites Perception Kinases, Genes involved in Entrapment OfPhosphatases, G- polar Auxin transport Microorganismal Proteins Genesinvolved in Symbionts Transcription starch deposition in Nutrient UptakeActivators the roots Synthesis Of Change In Genes involved inMetabolites And/Or Chromatin Structure production of reactive ProteinsAnd/Or Localized oxygen species Modulation Of DNA Topology Genesinvolved in Transduction Cell Wall Proteins flavonoid synthesis PathwaysCa⁺⁺ Fluctuation Specific Gene Reactive Oxygen Transcription Species(ROS) Initiation production Nutrient Uptake Enhancement Gravitropicgrowth of roots Associations with rhizobia are stimulated Down- GenesRepressed In Negative Transcription Regulated Root DevelopmentRegulation Of Factors Transcripts Responders To Primary Root, Kinases,Micro-Organismal Lateral Root, and/or Phosphatases, G- Symbionts AndRoot Hair Proteins Parasites Production Change In Genes With ReleasedChromatin Structure Discontinued Changes In And/Or DNA Expression OrPathways And Topology UnsTable mRNA In Processes Stability Of FactorsPresence Of Root Operating In Cells For Protein And/Or Micro- Changes InSynthesis And Organismal Metabolism Degradation Symbionts Inhibition ofroot Metabolic Enzymes gravitropism

Changes in the function or development of roots are the result ofmodulation of the activities of one or more of these many root genes andgene products. These genes and/or products are responsible for effectson traits such as plant vigor and seed yield, especially when plants aregrowing in the presence of soil borne biotic or abiotic stresses or whenthey are growing in barren conditions or in soils depleted of certainminerals.

Root genes, gene components and gene products can act alone or incombination as described in the introduction. Of particular interest arecombinations of these genes and gene products with those that modulatestress tolerance and/or metabolism. Stress tolerance and metabolismgenes and gene products are described in more detail in the sectionsbelow.

Use of Promoters of Root Genes

Promoters of root genes, as described in the Reference tables, forexample, can be used to modulate transcription that is induced by rootdevelopment or any of the root biological processes or activities above.For example, when a selected polynucleotide sequence is operably linkedto a promoter of a root gene, then the selected sequence is transcribedin the same or similar temporal, development or environmentally-specificpatterns as the root gene from which the promoter was taken. The rootpromoters can also be used to activate antisense copies of any codingsequence to achieve down regulation of its protein product in roots.They can also be used to activate sense copies of mRNAs by RNAinterference or sense suppression in roots.

III.A.2. Root Hair Genes, Gene Components and Products

Root hairs are specialized outgrowths of single epidermal cells termedtrichoblasts. In many and perhaps all species of plants, thetrichoblasts are regularly arranged around the perimeter of the root. InArabidopsis, for example, trichoblasts tend to alternate with non-haircells or atrichoblasts. This spatial patterning of the root epidermis isunder genetic control, and a variety of mutants have been isolated inwhich this spacing is altered or in which root hairs are completelyabsent.

III.A.2.a. Identification of Root Hair Genes

Root hair genes identified herein are defined as genes, gene componentsand products capable of modulating one or more processes in or thefunction of root hairs as described below. Root hairs are capable ofcontrolling or influencing many plant traits, also as shown below.Examples of such root hair development genes and gene products are shownin the Reference and Sequence Tables. The protein products of many ofthese genes are also identified in these Tables.

Root Hair Genes Identified by Differential Expression

These genes were discovered and characterized from a much larger set ofgenes by experiments designed to find genes whose mRNA products areassociated specifically with root hairs. These experiments made use ofthe arabidopsis mutant “root hairless” (rhl), which does not developroot hairs. By comparing gene expression profiles of rhl roots withthose of wild type roots grown in identical conditions, genesspecifically expressed in root hairs were revealed. The MA_diff Table(s)reports the transcript levels of the experiment (see EXPT ID: 108594,108433). For transcripts that had higher levels in the samples than thecontrol, a “+” is shown. A “−” is shown for when transcript levels werereduced in root tips as compared to the control. For more experimentaldetail see the Example section below.

Root Hairs genes are those sequences that showed differential expressionas compared to controls, namely those sequences identified in theMA_diff tables with a “+” or “−” indication.

Root Hairs Genes Identified by Cluster Analyses of DifferentialExpression

Root Hairs Genes Identified by Correlation to Genes that areDifferentially Expressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of Root Hairs genes is any group in the MA_clustthat comprises a cDNA ID that also appears in Expt ID 108594, 108433 theMA_diff table(s).

Root Hairs Genes Identified by Correlation to Genes that CausePhysiological Consequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of RootHairs genes. A group in the MA_clust is considered a Root Hairs pathwayor network if the group comprises a cDNA ID that also appears inKnock-in or Knock-out tables that causes one or more of the phenotypesdescribed in section above.

Root Hairs Genes Identified by Amino Acid Sequence Similarity

Root Hairs genes from other plant species typically encode polypeptidesthat share amino acid similarity to the sequences encoded by corn andArabidopsis Root Hairs genes. Groups of Root Hairs genes are identifiedin the Protein Group table. In this table, any protein group thatcomprises a peptide ID that corresponds to a cDNA ID member of a RootHairs pathway or network is a group of proteins that also exhibits RootHairs functions/utilities.

Examples of phenotypes, biochemical activities, and transcript profilesthat can be modulated by these genes and gene products are describedabove and below.

III.A.2.b. Use of Root Hair Development Genes to Modulate Phenotypes

The root hair development genes of the instant invention are useful tomodulate one or more processes of root hair structure and/or functionincluding (1) development; (2) interaction with the soil and soilcontents; (3) uptake and transport in the plant; and (4) interactionwith microorganisms.

1.) Development

The surface cells of roots can develop into single epidermal cellstermed trichoblasts or root hairs. Some of the root hairs will persistfor the life of the plant; others will gradually die back; some maycease to function due to external influences. The genes and geneproducts of this invention are useful to modulate any one or all ofthese growth and development process generally, as in root hair densityor root hair growth; including rate, timing, direction, and size, forexample. Processes that are regulated by these genes and gene productsinclude cell properties such as cell size, cell division, rate anddirection and number, cell elongation, cell differentiation, lignifiedcell walls, epidermal cells (including trichoblasts) and root apicalmeristem cells (growth and initiation); and root hair architecture suchas leaf cells under the trichome, cells forming the base of thetrichome, trichome cells, and root hair responses.

The genes and gene products of this invention are useful to modulate oneor more of the growth and development processes in response to internalplant programs or environmental stimuli in, for example, the seminalsystem, nodal system, hormone responses, Auxin, root cap abscission,root senescence, gravitropism, coordination of root growth anddevelopment with that of other organs (including leaves, flowers, seeds,fruits, and stems), and changes in soil environment (including water,minerals, Ph, and microfauna and flora).

2.) Interaction with Soil and Soil Contents

Root hairs are sites of intense chemical and biological activity and asa result can strongly modify the soil they contact. Roots hairs can becoated with surfactants and mucilage to facilitate these activities.Specifically, roots hairs are responsible for nutrient uptake bymobilizing and assimilating water, reluctant ions, organic and inorganiccompounds and chemicals. In addition, they attract and interact withbeneficial microfauna and flora. Root hairs also help to mitigate theeffects of toxic ions, pathogens and stress. Examples of root hairproperties and activities that the genes and gene products of theinvention are useful to modulate include root hair surfactant andmucilage (including composition and secretion rate and time); nutrientuptake (including water, nitrate and other sources of nitrogen,phosphate, potassium, and micronutrients (e.g. iron, copper, etc.);microbe and nematode associations (such as bacteria includingnitrogen-fixing bacteria, mycorrhizae, nodule-forming and othernematodes, and nitrogen fixation); oxygen transpiration; detoxificationeffects of iron, aluminum, cadium, mercury, salt, and other soilconstituents; pathogens (including chemical repellents) glucosinolates(GSL1), which release pathogen-controlling isothiocyanates; and changesin soil (such as Ph, mineral excess and depletion), and rhizosheath.

3.) Transport of Materials in Plants

Uptake of the nutrients by the root and root hairs contributes asource-sink effect in a plant. The greater source of nutrients, the moresinks, such as stems, leaves, flowers, seeds, fruits, etc. can drawsustenance to grow. Thus, root hair development genes and gene productsare useful to modulate the vigor and yield of the plant overall as wellas of distinct cells, organs, or tissues of a plant. The genes and geneproducts, therefore, can modulate Vigor, including plant nutrition,growth rate (such as whole plant, including height, flowering time,etc., seedling, coleoptile elongation, young leaves, stems, flowers,seeds and fruit) and yield, including biomass (fresh and dry weightduring any time in plant life, including maturation and senescence),number of flowers, number of seeds, seed yield, number, size, weight andharvest index (content and composition, e.g. amino acid, jasmonate, oil,protein and starch) and fruit yield (number, size, weight, harvestindex, and post harvest quality).

Additional Uses of Plants with Modified Root Hairs

Plants with root hairs modified in one or more of the propertiesdescribed above are used to provide:

-   -   A. Higher vigor and yield of plant and harvested products due to        pathogen resistance from conditioning the soil with        plant-derived chemicals and/or more tolerance to stresses such        as drought, flooding and anoxia    -   B. Better Animal (Including Human) Nutrition    -   C. Improved Dietary Mineral Nutrition    -   D. Increased Plant Survival By Decreasing Lodging    -   E. Better Plant Survival By:        -   (a) Decreased Lodging        -   (b) More Efficient Transport        -   (c) More Efficient Physiology        -   (d) More Efficient Metabolism    -   F. Increased Yield Of Valuable Molecules

Root Hair Modulation

To regulate any of the phenotype(s) above, activities of one or more ofthe root hair genes or gene products is modulated and tested byscreening for the desired trait. Specifically, the gene, mRNA levels, orprotein levels are altered in a plant utilizing the procedures describedherein and the phenotypes can be assayed. As an example, a plant can betransformed according to Bechtold and Pelletier (1998, Methods. Mol.Biol. 82:259-266) and/or screened for variants as in Winkler et al.(1998) Plant Physiol 118: 743-50 and visually inspected for the desiredphenotype or metabolically and/or functionally assayed according toDolan et al. (1993, Development 119: 71-84), Dolan et al. (1997,Development 124: 1789-98), Crawford and Glass (1998, Trends PlantScience 3: 389-95), Wang et al. (1998, PNAS USA 95: 15134-39), Gaxiolaet al. (1998, PNAS USA 95: 4046-50), Apse et al. (1999, Science 285:1256-58), Fisher and Long (1992, Nature 357: 655-60), Schneider et al.(1998, Genes Devel 12: 2013-21) and Hirsch (1999, Curr Opin Plant Biol.2: 320-326).

III.A.2.c. Use of Root Hair Development Genes to Modulate BiochemicalActivities

The activities of one or more of the root hair development genes can bemodulated to change biochemical or metabolic activities and/or pathwayssuch as those noted below. Such biological activities can be measuredaccording to the citations included in the table below:

Biochemical Or Metabolic Process Activities And/Or Pathways CitationsIncluding Assays Association Functions Associated Gage et al. (1996) JOf Root Hair With Root Hair Curling Bacteriol 178: 7159-66 With NitrogenAnd Signal Transduction Fixing Bacteria Root Hair Schneider et al.(1998) Spacing Genes Devel 12: 2013-21 Initiation Elongation MetabolismOrganic Molecule Export Moody et al. (1988) Phytochemistry 27: 2857-61Ion Export Uozumi et al. (2000) Plant Physiol 122: 1249-59 Frachisse etal. (2000) Plant J 21: 361-71 Nutrient Nutrient Uptake Frachisse et al.(2000) Plant Uptake J 21: 361-71 Uozumio et al. (2000) Plant Physiol122: 1249-59

Other biological activities that can be modulated by the root hair genesand gene products are listed in the Reference tables. Assays fordetecting such biological activities are described in the Protein Domaintable.

III.A.2.d. Use of Root Hair Genes, Gene Components and Product toModulate Transcription Levels

Many genes are “up regulated” or “down regulated” in root hairs orassociated with root hair formation because genes are regulated innetworks. Thus some root hairs genes are useful to regulate theactivities of many other genes, directly or indirectly to influencecomplex phenotypes. Examples of transcription profiles of root genes aredescribed in the Table below with associated biological activities. “Upregulated” profiles are those where the mRNA levels are higher when therhl gene is inhibited as compared to when rhl gene is not inhibited; andvice-versa for “down-regulated” profiles.

Transcript Physiological Examples Of Levels Type Of Genes ConsequencesBiochemical Activity Down Genes Expressed In Root Hair FormationTransporters Regulated Root Hair Microorganism Metabolic EnzymesTranscripts Development Perception Change In Cell Responders ToEntrapment Of Membrane Structure Micro-Organismal Microorganismal AndPotential Symbionts And Symbionts Kinases, Parasites Nutrient UptakePhosphatases, G- Synthesis Of Proteins Metabolites And/Or TranscriptionProteins Activators Modulation Of Change In Chromatin TransductionStructure And/Or Pathways Localized DNA Specific Gene TopologyTranscription Cell Wall Proteins Initiation Nutrient Uptake EnhancementUp-Regulated Genes Repressed In Negative Regulation TranscriptionFactors Transcripts Roots Making Of Hair Production Kinases, HairsReleased Phosphatases, G- Responders To Changes In ProteinsMicro-Organismal Pathways And Change In Chromatin Symbionts AndProcesses Operating Structure And/Or Parasites In Cells DNA TopologyGenes With Changes In Stability Of Factors Discontinued Metabolism ForProtein Synthesis Expression Or And Degradation UnsTable mRNAInMetabolic Enzymes Presence Of Root Cell Wall Proteins Hairs And/OrMicro-Organismal Symbionts

Changes in the patterning or development of root hairs are the result ofmodulation of the activities of one or more of these many root hairgenes and gene products. These genes and/or products are responsible foreffects on traits such as plant vigor and seed yield, especially whenplants are growing in the presence of biotic or abiotic stresses or whenthey are growing in barren conditions or in soils depleted of certainminerals.

Root hair genes and gene products can act alone or in combination asdescribed in the introduction. Of particular interest are combination ofthese genes and gene products with those that modulate stress toleranceand/or metabolism. Stress tolerance and metabolism genes and geneproducts are described in more detail in the sections below.

Use of Promoters of Root Hair Genes

Promoters of root hair development genes, as described in the Referencetables, for example, are useful to modulate transcription that isinduced by root hair development or any of the following phenotypes orbiological activities above. For example, any desired sequence can betranscribed in similar temporal, tissue, or environmentally-specificpatterns as the root hair genes when the desired sequence is operablylinked to a promoter of a root hair responsive gene.

III.A.3. Leaf Genes, Gene Components and Products

Leaves are responsible for producing most of the fixed carbon in a plantand are critical to plant productivity and survival. Great variabilityin leaf shapes and sizes is observed in nature. Leaves also exhibitvarying degrees of complexity, ranging from simple to multi-compound.Leaf genes as defined here, not only modulate morphology, but alsoinfluence the shoot apical meristem, thereby affecting leaf arrangementon the shoot, internodes, nodes, axillary buds, photosynthetic capacity,carbon fixation, photorespiration and starch synthesis. Leaf geneselucidated here can be used to modify a number of traits of economicinterest from leaf shape to plant yield, including stress tolerance, andto modify the efficiency of synthesis and accumulation of specificmetabolites and macromolecules.

III.A.3.a. Identification of Leaf Gene, Gene Components and Products

Leaf genes identified herein are defined as genes, active or potentiallyactive to greater extent in leaves than in some other organs of theplant or as genes that affect leaf properties. These genes and genecomponents are useful for modulating one or more processes in orfunctions of leaves, as described below, to improve plant traits rangingfrom yield to stress tolerance. Examples of such leaf genes and geneproducts are shown in the Reference and Sequence Tables and sequencesencoding polypeptides of the Protein Group and Protein Group Matrixtables or fragments thereof, Knock-In, Knock-Out and MA_diff Tables. Thebiochemical functions of the protein products of many of these genesdetermined from comparisons with known proteins are also given in theReference tables.

Leaf Genes Identified by Phenotypic Observations

Some leaf genes were discovered and characterized from a much larger setof genes by experiments designed to find genes that cause phenotypicchanges in leaf, petiole, internode, and cotyledon morphology.

In these experiments, leaf genes were identified by either (1) ectopicexpression of a cDNA in a plant or (2) mutagenesis of the plant genome.The plants were then cultivated and one or more of the following leafphenotypes, which varied from the parental “wild-type”, were observed:

A. Changes In Seedling Stage Cotyledons

-   -   Cup Shaped    -   Curled    -   Horizontally Oblong    -   Long Petioles    -   Short Petioles    -   Silver    -   Tricot    -   Wilted

B. Changes In Rosette And Flowering Stage Leaf Shapes

-   -   Cordate    -   Cup-Shaped    -   Curled    -   Fused    -   Lanceolate    -   Lobed    -   Long Petioles    -   Short Petioles    -   Oval    -   Ovate    -   Serrate    -   Trident    -   Undulate    -   Vertically Oblong

C. Changes In Cauline, Flowering Leaf Shape

-   -   Misshapen    -   Other

D. Changes In Leaf Pigment

-   -   Albino    -   Dark Green Pigment    -   High Anthocyanin    -   Interveinal Chlorosis    -   Yellow Pigment

E. Changes In Leaf Size

F. Changes In Seedling Stage Hypocotyl

-   -   Long    -   Short

G. Changes In Leaf Number

H. Changes In Wax Deposition

-   -   Glossy Rosette And Flowering Stage Leaves    -   Altered Wax Deposition On The Bolt

Leaf Genes Identified by Differential Expression

Also, leaf genes were identified in experiments in which theconcentration of mRNA products in the leaf, or stem, or Knock-out mutant3642-1 were compared with to a control. The MA_diff Table(s) reports thetranscript levels of the experiment (see EXPT ID: 108477, 108512,108497, 108498, 108598). For transcripts that had higher levels in thesamples than the control, a “+” is shown. A “−” is shown for whentranscript levels were reduced in root tips as compared to the control.For more experimental detail see the Example section below.

Leaf genes are those sequences that showed differential expression ascompared to controls, namely those sequences identified in the MA_difftables with a “+” or “−” indication.

Leaf Genes Identified by Cluster Analyses of Differential Expression

Leaf Genes Identified by Correlation to Genes that are DifferentiallyExpressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of Leaf genes is any group in the MA_clust thatcomprises a cDNA ID that also appears in Expt ID 108477, 108512, 108497,108498, 108598 of the MA_diff table(s).

Leaf Genes Identified by Correlation to Genes that Cause PhysiologicalConsequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of Leafgenes. A group in the MA_clust is considered a Leaf pathway or networkif the group comprises a cDNA ID that also appears in Knock-in orKnock-out tables that causes one or more of the phenotypes described insection above.

Leaf Genes Identified by Amino Acid Sequence Similarity

Leaf genes from other plant species typically encode polypeptides thatshare amino acid similarity to the sequences encoded by corn andArabidopsis Leaf genes. Groups of Leaf genes are identified in theProtein Group table. In this table, any protein group that comprises apeptide ID that corresponds to a cDNA ID member of a Leaf pathway ornetwork is a group of proteins that also exhibits Leaffunctions/utilities.

It is assumed that (i) the genes preferentially expressed in leaves areconcerned with specifying leaf structures and the synthesis of all theconstituent molecules and (ii) that the genes repressed in leavesspecify products that are not required in leaves or that could inhibitnormal leaf development and function.

Examples of phenotypes, biochemical activities, and transcriptionprofiles that are modulated by using selected members of these genes andgene products, singly or in combination, are described below.

III.A.3.b. Use of Leaf Genes, Genes Components and Products to ModulatePhenotypes

Leaves are critical for the performance and industrial utility ofplants. There is extensive evidence that the number, size, shape,position, timing of synthesis, timing of senescence and chemicalconstitution are very important for agriculture, horticulture and usesof plants as chemical factories for making valuable molecules. Manyimprovements already demonstrated over past decades have involvedgenetic modifications to leaves. Therefore, the leaf genes and genecomponents of this invention offer considerable opportunities forfurther improving plants for industrial purposes. When the leaf genesand/or gene components are mutated or regulated differently, they arecapable of modulating one or more of the processes determining leafstructure and/or function including (1) development; (2) interactionwith the environment and (3) photosynthesis and metabolism.

1.) Development

The leaf genes, gene components and products of the instant inventionare useful to modulate one or more processes of the stages of leafmorphogenesis including: stage 1—organogenesis that gives rise to theleaf primordium; stage 2—delimiting basic morphological domains; andstage 3—a coordinated processes of cell division, expansion, anddifferentiation. Leaf genes include those genes that terminate as wellas initiate leaf development. Modulating any or all of the processesleads to beneficial effects either at specific locations or throughoutthe plant, such as in the cotyledons, major leaves, cauline leaves, orpetioles.

Leaf genes, gene components and gene products are useful to modulatechanges in leaf cell size, cell division (rate and direction), cellelongation, cell differentiation, stomata size, number, spacing andactivity, trichome size and number, xylem and phloem cell numbers, cellwall composition, and all cell types. The leaf genese are also useful tomodulate to change overall leaf architecture, including veination (suchas improvements in photosynthetic efficiency, stress toleranceefficiency of solute and nutrient movement to and from the leaf areaccomplished by increases or decreases in vein placement and number ofcells in the vein); shape, either elongated versus rounded or symmetry,around either (e.g. abaxial-adaxial (dorsiventral) axis, apical-basal(proximodistal) axis, and margin-blade-midrib (lateral) axis; andbranching (improved plant performance to biotic and abiotic stress inheavy density planting is achieved by increases or decreases in leafbranch position or leaf branch length).

Shoot apical meristem cells differentiate to become leaf primordia thateventually develop into leaves. The genes, gene components and geneproducts of this invention are useful to modulate any one or all ofthese growth and development processes, by affecting timing and rate orplanes of cell divisions for example, in response to the internal plantstimuli and/or programs such as embryogenesis; germination; hormoneslike Auxin leaf senescence; phototropism; coordination of leaf growthand development with that of other organs (such as roots, flowers,seeds, fruits, and stems; and stress-related programs.

2.) Interaction with the Environment

Leaves are the main sites of photosynthesis and have various adaptationsfor that purpose. Flat laminae provide a large surface for absorbingsunlight; leaves are rich in chloroplasts and mitochondria; stomata inthe lower surface of the laminae allow gases to pass into and out of theleaves including water; and an extensive network of veins brings waterand minerals into the leaves and transports the sugar products producedby photosynthesis to the rest of the plant. examples of leaf propertiesor activities that are modulated by leaf genes, gene components andtheir products to facilitate interactions between a plant and theenvironment including pigment accumulation; wax accumulation on thesurface of leaves (e.g. improved protection of young leaves from waterborne pathogen attack such as downey mildew with increased waxproduction); oxygen gain/loss control; carbon dioxide gain/loss control;water gain/loss control; nutrient transport; light harvesting;chloroplast biogenesis; circadian rhythm control; light/dark adaptation;defense systems against biotic and abiotic stresses; metaboliteaccumulation; and secondary metabolite production in leaf mesophyl,epidermis and trichomes (such as increases in antifeeding secondarymetabolites such as strictosiden reduce herbivory and decreases insecondary metabolites improve plants as forage by reducing allergens orundigestible compounds).

3.) Photosynthesis and Metabolism

Many of the uses for plants depend on the success of leaves as thepowerhouses for plant growth, their ability to withstand stresses andtheir chemical composition. Leaves are organs with many different celltypes and structures. Most genes of a plant are active in leaves andtherefore leaves have very diverse of pathways and physiologicalprocesses. Pathways and processes that are modulated by leaf genes, genecomponents and products include photosynthesis, sugar metabolism, starchsynthesis, starch degradation, nitrate and ammonia metabolism, aminoacid biosynthesis, transport, protein biosynthesis, dna replication,repair, lipid biosynthesis and breakdown, protein biosynthesis, storageand breakdown, nucleotide transport and metabolism, cell envelopebiogenesis, membrane formation, mitochondrial and chloroplastbiogenesis, transcription and RNA metabolism, vitamin biosynthesis,steroid and terpenoid biosynthesis, devise secondary metabolitesynthesis, co-enzyme metabolism, flavonoid biosynthesis and degradation,synthesis of waxes, glyoxylate metabolism, and hormone perception andresponse pathways.

Uses of Plants that are Modified as Described Above

Altering leaf genes or gene products in a plant modifies one or moreplant traits, to make the plants more useful for specific purposes inagriculture, horticulture and for the production of valuable molecules.The modified plant traits include A higher yield of leaves and theirmolecular constituents (due to different number, size, weight, harvestindex, composition including and amounts and types of carbohydrates,proteins, oils, waxes, etc.; photosynthetic efficiency (e.g. reducedphotorespiration), absorption of water and nutrients to enhance yields,including under stresses such as high light, herbicides, and heat,pathways to accumulate new valuable molecules); more optimal leaf shapeand architecture—enhancing photosynthesis and enhancing appeal inornamental species (including size, number, pigment, and aroma; a betteroverall plant architecture—enhancing photosynthesis and enhancing appealin ornamental species petals, sepals, stamens, and carpels; better shadeavoidance for maximizing photosynthesis by, for example, altering leafplacement, to improve light capture and photosynthetic efficiency,thereby increasing yields; Reduced negative effects of high plantingdensity, by altering leaf placement to be more vertical instead ofparallel to the ground, for instance; More resistance to the deleteriouseffects of wind and mechanical damage; Better stress tolerance(including without limitation drought resistance, by decreasing waterloss, and pathogen resistance, including, for instance, insectresistance through internal insecticide levels and optimizing the leafshape to prevent runoff of insecticides); and better overall yield andvigor.

Plant yield of biomass and of constituent molecules and plant vigor aremodulated to create benefits by genetically changing the growth rate ofthe whole plant, (including height, flowering time, etc.), seedling,coleoptile elongation, young leaves flowers, seeds, and/or fruit, or bychanging the biomass, including fresh and dry weight during any time inplant life, (including maturation and senescence), number of flowers,seed yield including for example, number, size, weight, harvest index,content and composition (e.g. amino acid, jasmonate, oil, protein andstarch), and fruit yield (such as number, size, weight, harvest index,content and composition, e.g. amino acid, jasmonate, oil, protein andstarch).

To change any of the phenotype(s) in I, II, or III above, activities ofone or more of the leaf genes or gene products are modulated in anorganism and the consequence evaluated by screening for the desiredtrait. Specifically, the gene, mRNA levels, or protein levels arealtered in a plant utilizing the procedures described herein and thephenotypes can be assayed. As an example, a plant can be transformedaccording to Bechtold and Pelletier (Methods. Mol. Biol. 82:259-266(1998)) with leaf gene constructs and/or screened for variants as inWinkler et al., Plant Physiol. 118: 743-50 (1998) and visually inspectedfor the desired phenotype and metabolically and/or functionally assayedfor altered levels of relevant molecules.

III.A.3.c. Use of Leaf Genes Gene Components and Products to ModulateBiochemical Activities

Leaves are complex organs and their structure, function and propertiesresult from the integration of many processes and biochemicalactivities. Some of these are known from the published literature andsome can be deduced from the genes and their products described in thisapplication. Leaf genes, and gene components are used singly or incombination to modify these processes and biochemical activities andhence modify the phenotypic and trait characteristics described above.Examples of the processes and metabolic activities are given in theTable below. The resulting changes are measured according to thecitations included in the Table.

BIOCHEMICAL OR CITATIONS METABOLIC ACTIVITIES INCLUDING PROCESS AND/ORPATHWAYS ASSAYS Metabolism - anabolic Farnesylation Pei et al., Science282: 287-290 and catabolic Cell Wall Biosynthesis (1998); Cutler et al.,Nitrogen Metabolism Science 273: 1239 (1996) Secondary Metabolite Goupilet al., J Exptl. Botany Biosynthesis and 49: 1855-62 (1998) DegradationWalch-Liu et al., J Exppt. Botany 51, 227-237 (2000) Water ConservationAnd Stomatal Development And Allen et al., Plant Cell 11: Resistance ToDrought Physiology 1785-1798 (1999) And Other Related Production ofpolyols Li et al., Science 287: 300-303 Stresses Regulation of salt(2000) concentration Burnett et al., J Exptl. Botany ABA response(s) 51:197-205 (2000) Transport Anion and Ca2+ Accumulation Raschke, In:Stomatal Cation Fluxes K+ Fluxes Function, Zeiger et al. Eds., Na+Fluxes 253-279 (1987) Receptor - ligand binding Lacombe et al., PlantCell 12: Anion and Cation fluxes 837-51 (2000); Wang et al., PlantPhysiol. 118: 1421-1429 (1998); Shi et al., Plant Cell 11: 2393-2406(1999) Gaymard et al., Cell 94: 647-655 (1998) Jonak et al., Proc. Natl.Acad. Sci. 93: 11274-79 (1996); Sheen, Proc. Natl. Acad. Sci. 95: 975-80(1998); Allen et al., Plant Cell 11: 1785-98 (1999) Carbon FixationCalvin Cycle Wingler et al., Philo Trans R Photorespiration Soe Lond BBiol Sci 355, Oxygen evolution 1517-1529 (2000); RuBisCO Palecanda etal., Plant Mol Chlorophyll metabolism Biol 46, 89-97 (2001); ChloroplastBiogenesis and Baker et al., J Exp Bot 52, Metabolism 615-621 (2001)Fatty Acid and Lipid Chen et al., Acta Biochim Pol Biosynthesis 41,447-457 (1999) Glyoxylate metabolism Imlau et al., PlantCell II, 309-322Sugar Transport (1999) Starch Biosynthesis and Degradation HormonePerception and Hormone Receptors and Tieman et al., Plant J 26, 47-58Growth Downstream Pathways for (2001) ethylene Hilpert et al., Plant J26, 435-446 jasmonic acid (2001) brassinosteroid Wenzel et al., PlantPhys gibberellin 124, 813-822 (2000) Auxin Dengler and Kang, Curr Opincytokinin Plant Biol 4, 50-56 (2001) Activation Of Specific Tantikanjanaet al., Genes Kinases And Phosphatases Dev 15, 1577-1580 (2001)

Other biological activities that are modulated by the leaf genes andgene products are listed in the Reference tables. Assays for detectingsuch biological activities are described in the Protein Domain table,for example.

III.A.3.d. Use of Leaf Genes, Gene Components and Products to ModulateTranscription Levels

The expression of many genes is “upregulated” or downregulated” inleaves because some leaf genes and their products are integrated intocomplex networks that regulate transcription of many other genes. Someleaf genes, gene components and products are therefore useful formodifying the transcription of other genes and hence complex phenotypes,as described above. Profiles of leaf gene activities are described inthe Table below with associated biological activities. “Up-regulated”profiles are those where the mRNA transcript levels are higher in leavesas compared to the plant as a whole. “Down-regulated” profiles representhigher transcript levels in the whole plant as compared to leaf tissueonly.

EXAMPLES OF TYPE OF GENES PHYSIOLOGICAL BIOCHEMICAL WHOSE CONSEQUENCESOF ACTIVITIES OF GENE TRANSCRIPT TRANSCRIPTS MODIFYING GENE PRODUCTSWITH LEVELS ARE CHANGED PRODUCT LEVELS MODIFIED LEVELS Up RegulatedGenes Involved In Leaf Cells Transcription Transcripts Leaf CellProliferate And Factors, Signal Differentiation, Cell Differentiate;Transduction Division, Cell Proteins, Kinase Expansion And PhosphatasesGenes Involved In Leaf Structures Chromatin Positive Regulation Form AndExpand Remodeling Of Leaf Genes Hormone Repressors Of Root BiosynthesisAnd Other Non Leaf Enzymes Cell Types Receptors Genes Involved InPhotosynthesis And Light Harvesting Photosynthesis Plastid Coupled ToATP Differentiation Production Chlorophyll Biosynthesis Calvin CycleRibulose Activated Bisphosphate Chloroplast Carboxylase Biogenesis AndChloroplast Plastid Membranes Differentiation Synthesis ActivatedChloroplast Ribosome Biogenesis Other Genes Starch Biosynthesis StarchSynthase Involved In Lipid Biosynthesis Nitrate Reductase MetabolismNitrogen Terpenoid Metabolism —NO₃ Biosynthesis Reduced AndTranscription Amino Acids Made Factors Secondary TransportersMetabolites Kinases Produced Phosphatases And Signal TransductionProtein Chromatin Structure Modulators Down Genes Involved In Leaf GenesTranscription Regulated Negative Regulation Activated And Leaf FactorsGenes Of Leaf Genes Functions Induced; Signal Transduction Dark-AdaptedProteins - Kinases Metabolism And Phosphatases Suppressed MetabolicEnzymes Meristematic Genes Chromatin Suppressed Remodeling Proteins LeafMetabolic Pathways Induced

While leaf polynucleotides and gene products are used singly,combinations of these polynucleotides are often better to optimize newgrowth and development patterns. Useful combinations include differentleaf polynucleotides and/or gene products with a hormone responsivepolynucleotide. These combinations are useful because of theinteractions that exist between hormone-regulated pathways, nutritionalpathways and development.

Use of Leaf Gene Promoters

Promoters of leaf genes are useful for transcription of desiredpolynucleotides, both plant and non-plant. If the leaf gene is expressedonly in leaves, or specifically in certain kinds of leaf cells, thepromoter is used to drive the synthesis of proteins specifically inthose cells. For example, extra copies of carbohydrate transporter cDNAsoperably linked to a leaf gene promoter and inserted into a plantincrease the “sink” strength of leaves. Similarly, leaf promoters areused to drive transcription of metabolic enzymes that alter the oil,starch, protein, or fiber contents of a leaf. Alternatively, leafpromoters direct expression of non-plant genes that can, for instance,confer insect resistance specifically to a leaf. Additionally thepromoters are used to synthesize an antisense mRNA copy of a gene toinactivate the normal gene expression into protein. The promoters areused to drive synthesis of sense RNAs to inactivate protein productionvia RNA interference.

III.A.4. Trichome Genes and Gene Components

Trichomes, defined as hair-like structures that extend from theepidermis of aerial tissues, are present on the surface of mostterrestrial plants. Plant trichomes display a diverse set of structures,and many plants contain several types of trichomes on a single leaf. Thepresence of trichomes can increase the boundary layer thickness betweenthe epidermal tissue and the environment, and can reduce heat and waterloss. In many species, trichomes are thought to protect the plantagainst insect or pathogen attack, either by secreting chemicalcomponents or by physically limiting insect access to or mobility onvegetative tissues. The stellate trichomes of Arabidopsis do not have asecretory anatomy, but at a functional level, they might limit herbivoreaccess to the leaf in the field. In addition, trichomes are known tosecrete economically valuable substances, such as menthol in mintplants.

III.A.4.a. Identification of Trichome Genes, Gene Components andProducts

Trichome genes identified herein are defined as genes or gene componentscapable of modulating one or more processes in or functions of atrichome, as described below. These genes, their components and productsare useful for modulating diverse plant traits from production ofsecondary metabolites to pathogen resistance. Examples of such trichomegenes and gene products are shown in the Reference and Sequence Tablesand sequences encoding polypeptides of the Protein Group and ProteinGroup Matrix tables or fragments thereof, Knock-in, Knock-out, MA-diffand MA-clust. The biochemical functions of the protein products of manyof these genes determined from comparisons with known proteins are alsogiven in the Reference tables.

Trichome Genes Identified by Phenotypic Observation

Trichome genes were discovered and characterized from a much larger setof genes by experiments designed to find genes that cause phenotypicchanges in trichome number and morphology on leaf, internode, cotyledon,petiole, and inflorescence. In these experiments, trichome genes wereidentified by either (1) ectopic expression of a cDNA in a plant or (2)mutagenesis of the plant genome. The plants were then cultivated and oneor more of the following phenotypes, which varied from parental“wild-type”, were observed: (1) trichome number; (2) trichome spacing(clustering); or (3) trichome branching. The genes regulating trichomephenotypes are identified in the Knock-In and Knock-Out Tables.

Trichome Genes Identified by Differential Expression

Trichome genes were also discovered and characterized from a much largerset of genes by experiments designed to find genes whose mRNA productsare associated specifically or preferentially with trichomes. Theseexperiments made use of an Arabidopsis glaborous mutant and a hairymutant. By comparing gene expression profiles of the glabrous mutantwith those of the hairy mutant grown under identical conditions, genesspecifically or preferentially expressed in trichomes were revealed. TheMA_diff Table(s) reports the transcript levels of the experiment (seeEXPT ID: 108452). For transcripts that had higher levels in the samplesthan the control, a “+” is shown. A “−” is shown for when transcriptlevels were reduced in root tips as compared to the control. For moreexperimental detail see the Example section below.

Trichome genes are those sequences that showed differential expressionas compared to controls, namely those sequences identified in theMA_diff tables with a “+” or “−” indication.

Trichome Genes Identified by Cluster Analyses of Differential Expression

Trichome Genes Identified by Correlation to Genes that areDifferentially Expressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of Trichome genes is any group in the MA_clust thatcomprises a cDNA ID that also appears in Expt ID 108452 of the MA_difftable(s).

Trichome Genes Identified by Correlation to Genes that CausePhysiological Consequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of Trichomegenes. A group in the MA_clust is considered a Trichome pathway ornetwork if the group comprises a cDNA ID that also appears in Knock-inor Knock-out tables that causes one or more of the phenotypes describedin section above.

Trichome Genes Identified by Amino Acid Sequence Similarity

Trichome genes from other plant species typically encode polypeptidesthat share amino acid similarity to the sequences encoded by corn andArabidopsis Trichome genes. Groups of Trichome genes are identified inthe Protein Group table. In this table, any protein group that comprisesa peptide ID that corresponds to a cDNA ID member of a Trichome pathwayor network is a group of proteins that also exhibits Trichomefunctions/utilities.

It is assumed that the genes differentially expressed in trichomes orleaves producing trichomes are concerned with specifying trichomes andtheir functions and therefore modulations of such genes and theirproducts modify trichomes and their products.

Examples of phenotypes, biochemical activities, and transcriptionprofiles that can be modulated by selected numbers of these genes andgene products singly or in combinations are described above and below.

III.A.4.b. Use of Trichome Genes, Gene Components and Products toModulate Phenotypes

Trichome genes of the instant invention, when mutated or activateddifferently, are useful for modulating one or more processes of trichomestructure and/or function including: (1) development; (2) plant stresstolerance; and (3) biosynthesis or secretion of trichome-specificmolecules. Trichome genes, components and gene products are useful toalter or modulate one or more of the following phenotypes:

1.) Development

Trichome differentiation is integrated with leaf development, hormonelevels and the vegetative development phase. The first trichome at theleaf tip appears only after the leaf grows to ˜100 μm in length.Subsequent events proceed basipetally as the leaf grows. As leafdevelopment progresses, cell division patterns become less regular andislands of dividing cells can be observed among differentiated pavementcells with their characteristic lobed morphology. Trichome initiation inthe expanding leaf occurs within these islands of cells and oftendefines points along the perimeter of a circle, with an existingtrichome defining the center.

Once a cell enters the trichome pathway it undergoes an elaboratemorphogenesis program that has been divided into different stages basedon specific morphological hallmarks. The trichome genes, gene componentsand gene products of this invention are useful to modulate any one orall of these growth and development processes by affecting rate, timing,direction and size, for example. Trichome genes can also affect trichomenumber and the organs on which they occur, type of trichomes such asglandular trichomes and stellate trichomes; cell properties such as cellsize, cell division rate and direction, cell elongation, celldifferentiation, secretory cells, trichome number (average trichomenumber per leaf for mint: 13,500,000), cell walls, cell death, andresponse to reactive oxygen species; trichome architecture such astrichome cell structure, placement on leaf, and secretory systems; andtrichome responses. Trichome genes, gene components and gene products ofthis invention are useful to modulate one or more of the growth anddevelopment processes above; as in timing and rate, for example. Inaddition, the polynucleotides and polypeptides of the invention cancontrol the response of these processes to internal plant programs andsignaling molecules such as leaf development, hormones (includingabscisic acid, Auxin, cytokinin, gibberellins, and brassinosteroids,apoptosis; and coordinated trichome growth and development in flowers,stems, petioles, cotyledons, and hypocotyls.

2.) Plant Stress Tolerance

The physical characteristics of trichomes as well as the substancessecreted by trichomes are useful in protecting the plant from bothbiotic and abiotic attacks. Thus, selected trichome genes and geneproducts can be used to help protect distinct cells, organs, or tissuesas well as overall plant yield and vigor. Examples of stresses,tolerances to which are modulated by trichome genes and gene productsare drought (e.g., trichome number variation can decrease the surfacearea that allows evaporation), heat (e.g., trichomes can produce shadeand provide protection for meristems), salt, insects (e.g., trichomescan prevent insects from settling on plant surfaces), herbivory (e.g.,trichomes can produce harmful chemicals), and ultraviolet light.

3.) Biosynthesis, Accumulation or Secretion of Metabolites

The glandular trichomes from various species are shown to secrete and,sometimes, locally synthesize a number of substances including salt,monoterpenes and sesquiterpenes, terpenoids, exudate, insect entrappingsubstances, antifeedants, pheromones, and others. Therefore, trichomegenes can be used to modulate the synthesis, accumulation and secretionof a large number of metabolites especially related to trichome biology.Some are synthesized in response to biotic and abiotic stresses. For amore detailed description of these metabolites see the section “Use ofTrichome Genes to Modulate Biochemical Activities” below.

Uses of Plants that are Modified as Described Above

Altering trichome properties is useful for modifying one or more planttraits making the plants more useful in agriculture, horticulture andfor the production of valuable molecules. These plant traits includeProduction of specific carbohydrates, proteins, oils, aromas, flavors,pigments, secondary metabolites such as menthol (and othermonoterpenes), etc., that can be used in situ or purified and used in awide variety of industries; Increased production of moleculessynthesized in trichomes by increasing the trichome number on differentplant organs, such as cotyledons, leaves, hypocotyls, stems, petioles,etc.; Increased cotton fibers per boll due to decreased numbers oftrichomes that reduces insect hiding and contamination; More optimalgrowth rate of a whole plant or specific parts of a plant due to moreoptimal trichome cellular development and the better resistance tobiotic/abiotic stresses (including plant parts such as whole plantseedling, coleoptile elongation, young leaves, flowers, seeds, andfruit); increased harvested yield of plants, organs and theirconstituent molecules including biomass (such as fresh and dry weightduring any time in plant life, including maturation and senescence,number of flowers, seed yield in terms of number, size, weight, harvestindex, content and composition, e.g. amino acid, jasmonate, oil, proteinand starch, and fruit yield in terms of number, size, weight, harvestindex, post harvest quality, content and composition, e.g. amino acid,jasmonate, oil, protein and starch).

To regulate any of the phenotype(s) above, activities of one or more ofthe trichome genes or gene products can be modulated in an organism andtested by screening for the desired trait. Specifically, the gene, mRNAlevels, or protein levels can be altered in a plant utilizing theprocedures described herein and the phenotypes can be assayed. As anexample, a plant can be transformed according to Bechtold and Pelletier(Methods. Mol. Biol. 82:259-266 (1998)) and/or screened for variants asin Winkler et al., Plant Physiol. 118: 743-50 (1998) and visuallyinspected for the desired phenotype or metabolically and/or functionallyassayed.

III.A.4.c. Use of Trichome Genes, Gene Components and Products toModulate Biochemical Activities

The phenotype traits outlined above result from the integration of manycellular trichome associated processes and biochemical activities. Someof these are known from published literature and some can be deducedfrom the genes discovered in the MA Tables, etc. One or more of thesetrichome genes, gene components and products are useful to modulatethese cellular processes, biochemical or metabolic activities and/orpathways such as those noted below. Such biological activities can bemeasured according to the citations included in the table below:

BIOCHEMICAL OR METABOLIC ACTIVITIES AND/OR CITATIONS INCLUDING PROCESSPATHWAYS ASSAYS Growth, Cell wall biosynthetic Molhoj et. al. (2001).Plant Differentiation enzymes Mol. Biol. 46, 263-275 And DevelopmentCell fate determination Krishnakumar and proteins Oppenheimer (1999).Major pathways of carbon Development 1221, 3079-3088. and nitrogenmetabolism Kroumova et al. (1994). PNAS 91, 11437-11441 WaterCytoskeleton and Trichome Schnittger et al. (1999). Conservation Andmorphology and spacing Plant Cell 11, 1105-1116 Resistance To controlsHulskamp et al (1994). Cell Drought And 76, 555-566 Other RelatedStresses Trichome exudate Insect repellant Insects and The PlantSurface, pp 151-172, Edward Arnold, London (1986) Terpenoid Terpenoidbiosynthesis Alonso et al. (1992). J. Biol. biosynthesis enzymesincluding: Chem. 267, 7582-7587 including FarnesyltranstransferaseRajonarivony et al (1992). monoterpenes and Geranylgeranyl- Arch.Biochem. Biophys. sesquiterpenes diphosphate synthase 299, 77-82Geranyltranstransferase Farnesyl-diphosphate synthaseDimethylallyltranstransferase Geranyl-diphosphate synthase H₂O₂ NADPHoxidase (subunit) Alverez et al (1998) Cell 92, accumulation andsynthesis and function 773-784 activation of SAR Grant Orozco-Cardenasand Ryan (1999) PNAS 96, 6553-6557 Antifeedants Lactone biosynthesisParuch et al. (2000). J. biosynthesis and enzymes Agric. Food Chem. 48,secretion 4973-4977 Pheromone Farnesine biosynthesis Teal et al. (1999)Arch. biosynthesis and enzymes Insect Biochem Physiol. 42, secretion225-232 Endoreplication Cyclin and cyclin dependant De Veylder et al.(2001) kinases Plant Cell 13, 1653-1668 De Veylder et al. (2001) PlantJ. 25, 617-626

Specific enzyme and other activities associated with the functions ofindividual trichome genes that can be modulated by the trichome genesand gene products are listed in the Reference tables where the functionsof individual genes and their products are listed. Assays for detectingsuch biological activities are described in the Protein Domain table,for example.

III.A.4.d. Use of Trichome Genes, Gene Components and Products toModulate Phenotypes by Modulating Transcription Levels of Other Genes

Many of the genes are “up regulated” or “down regulated” in trichomesbecause they are regulated as members of networks or cascade of genesunder the control of regulatory genes. Thus some trichome genes areuseful to influence levels of other genes and so orchestrate the complexphenotypes. Examples of the types of genes with altered transcriptlevels in trichomes are described in the Table below, together withassociated biological activities. “Up-regulated” profiles are thosewhere the mRNA levels are higher in the glaborous plants as compared tothe “hairy” plant. “Down-regulated” profiles represent higher transcriptlevels in the “hairy” plant as compared to the glaborous plant.

PHYSIOLOGICAL EXAMPLES OF TYPE OF GENES CONSEQUENCES BIOCHEMICAL WHOSEOF MODIFYING ACTIVITIES WHOSE TRANSCRIPT TRANSCRIPTS ARE GENE PRODUCTTRANSCRIPTS ARE LEVELS CHANGED LEVELS CHANGED Up Regulated Genes activein Changes in Transcription Transcripts suppressing trichome HormoneFactors formation Perception Transporters Changes in Change In Cell G-Hormone proteins Biosynthesis Kinases And Changes in PhosphatasesSpecific Gene Transcription Transcription factors Initiation Ca-bindingproteins Changes in Transcription cytoskeleton and Activators cell wallChange In assembly and Chromatin structure Structure And/Or LocalizedDNA Topology Specific Factors (Initiation And Elongation) For ProteinSynthesis Maintenance Of mRNA Stability Maintenance Of Protein StabilityMaintenance Of Protein-Protein Interaction Down-Regulated Genes activein Changes in Transcription Transcripts inducing formation of HormoneFactors trichomes Perception Change In Protein Changes in Structure ByHormone Phosphorylation Biosynthesis (Kinases) Or Changes inDephosphorylation Specific Gene (Phosphatases) Transcription Change InInitiation Chromatin Changes in Structure And/Or cytoskeleton and DNATopology cell wall G-proteins, Ca2+- assembly and binding proteinsstructure Genes associated with Changes in cell Trichome size, cellshape differentiation and structure Genes associated with Changes intrichome-specific terpenoid metabolic pathways biosynthesis Changes inantifeedant and pheromone biosynthesis

While trichome polynucleotides and gene products can act alone,combinations of these polynucleotides also affect growth, developmentand leaf biochemistry. Combinations of trichome polynucleotide(s) and/orgene product(s) with genes or gene products involved in leafdevelopment, hormone responses, or vegetative development are usefulbecause trichome development is integrated with these processes.

Use of Promoters of Trichome Genes

Promoters of trichome genes are useful for facilitating transcription ofdesired polynucleotides, both plant and non-plant in trichomes. Forexample, extra copies of existing terpenoid synthesis coding sequencescan be operably linked to a trichome gene promoter and inserted into aplant to increase the terpenoids in the trichome. Alternatively,trichome promoters can direct expression of non-plant genes or genesfrom another plant species that can, for instance, lead to newterpenoids being made. The promoters can also be operably linked toantisense copies of coding sequences to achieve down regulation of thesegene products in cells.

III.A5. Chloroplast Genes, Gene Components and Products

The chloroplast is a complex and specialized organelle in plant cells.Its complexity comes from the fact that it has at least sixsuborganellar compartments subdivided by double-membrane envelope andinternal thylakoid membranes. It is specialized to carry out differentbiologically important processes including photosynthesis and amino acidand fatty acid biosynthesis. The biogenesis and development ofchloroplast from its progenitor (the proplasptid) and the conversion ofone form of plastid to another (e.g., from chloroplast to amyloplast)depends on several factors that include the developmental andphysiological states of the cells.

One of the contributing problems that complicate the biogenesis ofchloroplast is the fact that some, if not most, of its components mustcome from the outside of the organelle itself. The import mechanismsmust take into account to what part within the differentsub-compartments the proteins are being targeted; hence the proteinsbeing imported from the cytoplasm must be able to cross the differentinternal membrane barriers before they can reach their destinations. Theimport mechanism must also take into account how to tightly coordinatethe interaction between the plastid and the nucleus such that bothnuclear and plastidic components are expressed in a synchronous andorchestrated manner. Changes in the developmental and physiologicalconditions within or surrounding plant cells can consequently changethis tight coordination and therefore change how import mechanisms areregulated as well. Manipulation of these conditions and modulation ofexpression of the import components and their function can have criticaland global consequences to the development of the plant and to severalbiochemical pathways occurring outside the chloroplast. Expressionpatterns of such genes have been determined using microarray technology.

Microarray technology allows monitoring of gene expression levels forthousands of genes in a single experiment. This is achieved byhybridizing labeled fluorescent cDNA pools to glass slides that containspots of DNA (Schena et al. (1995) Science 270: 467-70). The USArabidopsis Functional Genomics Consortium (AFGC) has recently madepublic the results from such microarray experiments conducted with AFGCchips containing about 10,000 non-redundant ESTs, selected from about37,000 randomly sequenced ESTs generated from mRNA of different tissuesand developmental stages.

The sequences of the ESTs showing at least two-fold increases ordecreases in a mutant in a mutant (CiA2) of Arabidopsis thaliana, thatis distributed in chloroplast biogenesis relative to wild type grown inthe same conditions were identified, compared to the Ceres full lengthcDNA and genomic sequence databanks, and equivalent Ceres clonesidentified. The MA_diff table reports the results of this analysis,indicating those Ceres clones which are up or down regulated overcontrols, thereby indicating the Ceres clones that are involved in theimport of proteins to chloroplast and chloroplast biogenesis.

Examples of genes and gene products that are involved in the import ofproteins to chloroplast are shown in the Reference, Sequence, ProteinGroup, and Protein Group Matrix tables. While chloroplast protein importpolynucleotides and gene products can act alone, combinations of thesepolynucleotides also affect growth and development. Useful combinationsinclude different chloroplast protein import responsive polynucleotidesand/or gene products that have similar transcription profiles or similarbiological activities, and members of the same or functionally relatedbiochemical pathways. Whole pathways or segments of pathways arecontrolled by transcription factor proteins and proteins controlling theactivity of signal transduction pathways. Manipulation of one or morechloroplast protein import gene activities are useful to modulate thebiological processes and/or phenotypes listed below. Chloroplast proteinimport responsive genes and gene products can act alone or incombination. Useful combinations include chloroplast protein importresponsive genes and/or gene products with similar transcriptionprofiles, similar biological activities, or members of the same orfunctionally related biochemical pathways. Here, in addition topolynucleotides having similar transcription profiles and/or biologicalactivities, useful combinations include polynucleotides that may havedifferent transcription profiles but which participate in common oroverlapping pathways. Whole pathways or segments of pathways arecontrolled by transcription factor proteins and proteins controlling theactivity of signal transduction pathways. Therefore, manipulation ofsuch protein levels is especially useful for altering phenotypes andbiochemical activities of plants. Manipulation of one or morechloroplast protein import gene activities are useful to modulate thebiological processes and/or phenotypes listed below.

Such chloroplast protein import responsive genes and gene products canfunction to either increase or dampen the above phenotypes or activitiesin response to changes in the regulation of import mechanisms. Further,promoters of chloroplast protein transport responsive genes, asdescribed in the Reference tables, for example, are useful to modulatetranscription that is induced by chloroplast protein transport or any ofthe following phenotypes or biological activities below. Further, anydesired sequence can be transcribed in similar temporal, tissue, orenvironmentally specific patterns as the chloroplast protein transportresponsive genes when the desired sequence is operably linked to apromoter of a chloroplast protein transport responsive gene. The MA_diffTable(s) reports the transcript levels of the experiment (see EXPT ID:Chloroplast (relating to SMD 8093, SMD 8094)). For transcripts that hadhigher levels in the samples than the control, a “+” is shown. A “−” isshown for when transcript levels were reduced in root tips as comparedto the control. For more experimental detail see the Example sectionbelow.

Chloroplast genes are those sequences that showed differentialexpression as compared to controls, namely those sequences identified inthe MA_diff tables with a “+” or “−” indication.

Chloroplast Genes Identified by Cluster Analyses of DifferentialExpression Chloroplast

Genes Identified by Correlation to Genes that are DifferentiallyExpressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of Chloroplast genes is any group in the MA_clustthat comprises a cDNA ID that also appears in Expt ID Chloroplast(relating to SMD 8093, SMD 8094) of the MA_diff table(s).

Chloroplast Genes Identified by Correlation to Genes that CausePhysiological Consequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks ofChloroplast genes. A group in the MA_clust is considered a Chloroplastpathway or network if the group comprises a cDNA ID that also appears inKnock-in or Knock-out tables that causes one or more of the phenotypesdescribed in section above.

Chloroplast Genes Identified by Amino Acid Sequence Similarity

Chloroplast genes from other plant species typically encode polypeptidesthat share amino acid similarity to the sequences encoded by corn andArabidopsis Chloroplast genes. Groups of Chloroplast genes areidentified in the Protein Group table. In this table, any protein groupthat comprises a peptide ID that corresponds to a cDNA ID member of aChloroplast pathway or network is a group of proteins that also exhibitsChloroplast functions/utilities.

III.A.5.a. Use of Chloroplast Protein Import Responsive Genes toModulate Phenotypes

Chloroplast protein import responsive genes and gene products are usefulto or modulate one or more phenotypes, including growth, roots, stems,and leaves; development, including plastid biogenesis, plastid division,plastid development and thylakoid membrane structures differentiationincluding plastid/chloroplast differentiation; photosynthesis includingcarbon dioxide fixation; transport including transcription/translationregulation within transport complex, phosphate translocation, andtargeted starch deposition and accumulation; and biosynthesis ofessential compounds such as lipid biosynthesis, riboflavin biosynthesis,carotenoid biosynthesis, and aminoacid biosynthesis.

To improve any of the phenotype(s) above, activities of one or more ofthe chloroplast protein import responsive genes or gene products can bemodulated and the plants tested by screening for the desired trait.Specifically, the gene, mRNA levels, or protein levels can be altered ina plant utilizing the procedures described herein and the phenotypes canbe assayed. As an example, a plant can be transformed according toBechtold and Pelletier (1998, Methods. Mol. Biol. 82:259-266) and/orscreened for variants as in Winkler et al. (1998) Plant Physiol 118:743-50 and visually inspected for the desired phenotype or metabolicallyand/or functionally assayed according to Saito et al. (1994, PlantPhysiol. 106: 887-95), Takahashi et al (1997, Proc. Natl. Acad. Sci. USA94: 11102-07) and Koprivova et al. (2000, Plant Physiol. 122: 737-46).

III.A.5.b. Use of Chloroplast Protein Import-Responsive Genes toModulate Biochemical Activities

The activities of one or more of the chloroplast protein importresponsive genes can be modulated to change biochemical or metabolicactivities and/or pathways such as those noted below. Such biologicalactivities can be measured according to the citations included in thetable below:

BIOCHEMICAL OR CITATIONS METABOLIC ACTIVITIES INCLUDING GENERAL CATEGORYAND/OR PATHWAYS ASSAYS Cell Growth and Regulation of Leaf Reinbothe etal. (1997) Proc. Differentiation Development Including Natl. Acad. Sci.USA. Photosynthetic 94: 8890-8894 Apparatus Eggink and Hoober (2000) J.Biol. Chem. 275: 9087-9090 Jagtap et al. (1998) J Exptl Botany 49:1715-1721 Regulation of Plastid Lawrence and Kindle (1997) Biogenesisand Plastid J. Biol. Chem. 272: 20357-20363 Division Lahiri and Allison(2000) Plant Physiol. 123: 883-894 Development of Plastid Kouranov etal. (1999) J. Inner/Outer and Biol. Chem. 274: 25181-25186 thylakoidMembrane Jackson et al. (1998) J. Biol. Structures Chem. 273:16583-16588 Li and Chen (1997) J. Biol. Chem. 272: 10968-10974 Lawrenceand Kindle (1997) J. Biol. Chem. 272: 20357-20363 Silva-Filho et al.(1997) J. Biol. Chem. 272: 15264-15269 Regulation of May and Soll (2000)Plant transcription and/or Cell 12: 53-63 translation related to Caliebeet al. (1997) EMBO maintenance of stability J. 16: 7342-7350 ofprotein-protein interaction within transport complex PhysiologyModulation of Sung and Krieg (1979) Plant Photosynthesis Physiol 64:852-56 Regulation of Lipid Bourgis et al. (1999) Plant BiosynthesisPhysiol. 120: 913-922 Reverdatto et al. (1999) Plant Physiol. 119:961-978 Roesler et al. (1997) Plant Physiol. 113: 75-81 Regulation ofRiboflavin Jordan et al. (1999) J. Biol. (Vitamin B) biosynthesis Chem.274: 22114-22121 Regulation of phosphate Flugge (1999) Annu. Rev.translocation across Plant Physiol. Plant Mol. chloroplast membraneBiol. 50: 27-45 Silva-Filho et al. (1997) J. Biol. Chem. 272:15264-15269 Regulation of targeted Yu et al. (1998) Plant starchdepostion and Physiol. 116: 1451-1460 accumulation Modulation of proteinSummer and Cline (1999) targeting and Plant Physiol. 119: 575-584translocation across Dabney-Smith et al. (1999) chloroplast membrane J.Biol. Chem. 274: 32351-32359 Hinnah et al. (1997) EMBO J. 16: 7351-7360Regulation of carotenoid Bonk et al. (1996) Plant biosynthesis Physiol.111: 931-939 Regulation of amino acid Flugge (1999) Annu. Rev.biosynthesis Plant Physiol. Plant Mol. Biol. 50: 27-45 Regulation ofsecondary Flugge (1999) Annu. Rev. metabolism Plant Physiol. Plant Mol.Biol. 50: 27-45 Signal Transduction Regulation of gene Chen et al.(2000) Plant transcriptional activity Physiol. 122: 813-822. specific tochloroplast Macasev et al. (2000) Plant protein import Physiol. 123:811-816 Regulation of protein Lang et al. (1998) J. Biol. target signalcleavage Chem. 273: 30973-30978 and protein degradation Jackson et al.(1998) J. Biol. Chem. 273: 16583-16588 Richter and Lamppa (1998) Proc.Natl. Acad. Sci. USA. 95: 7463-7468 Regulation of ion Van der Wijngaardand channel conformation Vredenberg (1999) J. Biol. and activity Chem.274: 25201-25204 Regulation of kinase and Waegemann and Soll (1996)phosphatases synthesis J. Biol. Chem. 271: 6545-6554 and activity Li etal. (2000) Science 287-300-303 Muller et al. (2000) J. Biol. Chem. 275:19475-19481 Modulation of Molecular Bonk et al. (1996) Plant Chaperoneand Other Physiol. 111: 931-939 Protein Folding Activity Walker et al.(1996) J. Biol. Chem. 271: 4082-4085 Kessler and Blobel (1996). Proc.Natl. Acad. Sci. USA 93: 7684-7689 Jackson et al. (1998) J. Biol. Chem.273: 16583-16588

Other biological activities that can be modulated by the chloroplastprotein import responsive genes and gene products are listed in theReference tables. Assays for detecting such biological activities aredescribed in the Protein Domain table.

Chloroplast protein import responsive genes are characteristicallydifferentially transcribed in response to fluctuating chloroplastprotein import levels or concentrations, whether internal or external toan organism or cell. The MA_diff reports the changes in transcriptlevels of various chloroplast protein import responsive genes that aredifferentially expressed among the mutants and the wild type.

Profiles of some of these chloroplast protein import responsive genesare shown in the Table below together with examples of the kinds ofassociated biological activities.

EXAMPLES OF TRANSCRIPT PHYSIOLOGICAL BIOCHEMICAL LEVELS TYPE OF GENESCONSEQUENCES ACTIVITY Up regulated Responders to ChloroplastTransporters transcripts defective chloroplast protein import Metabolicenzymes protein import regulation Change in cell Genes induced byChloroplast membrane structure defective import protein import and andpotential transport Kinases and Chloroplast phosphatases importTranscription metabolism activators Synthesis of Change in secondarychromatin structure metabolites and/or and/or localized proteins DNAtopology Modulation of Redox control chloroplast import Metabolicenzymes response concerned with transduction chloroplast pathwaysbiochemistry Changes in Organelle gene chloroplast expression andmembranes translation Specific gene transcription initiation Chloroplastand non-chloroplast metabolic pathways Down-regulated Responders toRegulation of Transcription transcripts defective chloroplastchloroplast protein factors protein import. import pathways Change inprotein Genes repressed by released structure by defective chloroplastChloroplast phosphorylation protein import protein import and (kinases)or Genes with unsTable transport dephosphoryaltion mRNAs whenChloroplast (phosphatases) chloroplast import is import Change indefective metabolism chromatin structure Genes with Changes in and/orDNA discontinued pathways and topology expression or processes Stabilityfactors for unsTable mRNA in operating in protein mRNA presence ofchloroplasts synthesis and chloroplast protein Changes in degradationimport organelle Organelle membranes transcription and Loss of organelletranslation proteins gene expression, Metabolic enzymes RNA and proteinsynthesis Changes in metabolism other than chloroplast protein importpathways Chloroplast import metabolism

Use of Promoters of Chloroplast Genes

Promoters of Chloroplast genes are useful for transcription of anydesired polynucleotide or plant or non-plant origin. Further, anydesired sequence can be transcribed in a similar temporal, tissue, orenvironmentally specific patterns as the Chloroplast genes, where thedesired sequence is operably linked to a promoter of a Chloroplast gene.The protein product of such a polynucleotide is usually synthesized inthe same cells, in response to the same stimuli as the protein productof the gene from which the promoter was derived. Such promoter are alsouseful to produce antisense mRNAs to down-regulate the product ofproteins, or to produce sense mRNAs to down-regulate mRNAs via sensesuppression

III.A.6. Reproduction Genes, Gene Components and Products

Reproduction genes are defined as genes or components of genes capableof modulating any aspect of sexual reproduction from flowering time andinflorescence development to fertilization and finally seed and fruitdevelopment. These genes are of great economic interest as well asbiological importance. The fruit and vegetable industry grosses over $1billion USD a year. The seed market, valued at approximately $15 billionUSD annually, is even more lucrative.

Expression of many reproduction genes and gene products is orchestratedby internal programs or the surrounding environment of a plant, asdescribed below. These genes and/or products have great importance indetermining traits such as fruit and seed yield. Examples of suchreproduction genes and gene products are shown in the Reference,Sequence, Protein Group, Protein Group Matrix tables, Knock-in,Knock-out, MA-diff and MA-clust. The biochemical functions of theprotein products of many of these genes determined from comparisons withknown proteins are also given in the Reference tables.

Reproduction Genes Identified by Phenotypic Observation

Reproduction genes were discovered and characterized from a much largerset of genes by experiments designed to find genes that cause phenotypicchanges in flower, silique, and seed morphology. In these experiments,reproduction genes were identified by either (1) ectopic expression of acDNA in a plant or (2) mutagenesis of the plant genome. The plants werethen cultivated and phenotypes, which varied from the parental“wild-type”, were observed.

One particular example of reproductive genes are those that areregulated by AP2. AP2 is a transcription factor that regulates manygenes, both as a repressor of some genes and as an activator of others.Some of these genes are those which establish the floral meristem orthose which regulate floral organ identity and development. As such, AP2has an effect on reproduction. This is, loss of AP2 activity iscorrelated with decreased male and female reproduction. AP2 is alsoknown to have an effect on seed mass, and therefore on yield. That is,overexpression of AP2 is correlated with smaller seeds or seedless fruitwhile repression of AP2 correlates with larger seeds (see, e.g. U.S.Pat. No. 5,994,622).

Another example of reproduction genes are those that are regulated byPISTILLATA (PI). PI is a transcription factor that regulates many genesboth as a repressor and activator. Some of these genes are those whichregulate floral organ identity and development, in conjunction withother transcription factors such as AP2 and AGAMOUS. As such, PI has aneffect on reproduction in that loss of PI activity is correlated withdecreased male reproduction. PI is also known to have an effect oncarpel number, and therefore potentially on ovule/seed number and yield.Specifically, repression of PI results in increased carpel number andtherefore ovule number.

Yet another example of reproductive genes are those that are regulatedby MEDEA (MEA). MEA is a SET-domain containing protein that associateswith other proteins to form complexes that affect chromatin structureand therefore gene expression. As such, loss of MEA function iscorrelated with global gene activation and repression leading to manyphenotypes including decreased female reproduction and therefore reducedseed set and yield.

In the characterization of these and other reproduction genes, thefollowing phenotypes were scored:

I. Flower

-   -   Size        -   Large        -   Small    -   Shape        -   Abnormal organ numbers        -   Agamous        -   AP-2 like    -   Color    -   Number    -   Fused Sepals

II. Silique

-   -   Size    -   Seed number        -   Reduced        -   Absent    -   Seed color

The identified genes regulating reproduction are identified in theKnock-in and Knock-out Tables.

Reproduction Genes Identified by Differential Expression

Reproduction genes were also identified in experiments designed todiscover genes whose mRNA products were in different concentrations inwhole flowers, flower parts, and siliques relative to the plant as awhole. The MA_diff Table(s) reports the transcript levels of theexperiment (see EXPT ID: 108473, 108474, 108429, 108430, 108431, 108475,108476, 108501). For transcripts that had higher levels in the samplesthan the control, a “+” is shown. A “−” is shown for when transcriptlevels were reduced in root tips as compared to the control. For moreexperimental detail see the Example section below.

Reproduction genes are those sequences that showed differentialexpression as compared to controls, namely those sequences identified inthe MA_diff tables with a “+” or “−” indication.

Reproduction Genes Identified by Cluster Analyses of DifferentialExpression

Reproduction Genes Identified by Correlation to Genes that areDifferentially Expressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of Reproduction genes is any group in the MA_clustthat comprises a cDNA ID that also appears in Expt ID 108473, 108474,108429, 108430, 108431, 108475, 108476, 108501 of the MA_diff table(s).

Reproduction Genes Identified by Correlation to Genes that CausePhysiological Consequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks ofReproduction genes. A group in the MA_clust is considered a Reproductionpathway or network if the group comprises a cDNA ID that also appears inKnock-in or Knock-out tables that causes one or more of the phenotypesdescribed in section above.

Reproduction Genes Identified by Amino Acid Sequence Similarity

Reproduction genes from other plant species typically encodepolypeptides that share amino acid similarity to the sequences encodedby corn and Arabidopsis Reproduction genes. Groups of Reproduction genesare identified in the Protein Group table. In this table, any proteingroup that comprises a peptide ID that corresponds to a cDNA ID memberof a Reproduction pathway or network is a group of proteins that alsoexhibits Reproduction functions/utilities.

It is assumed that the reproduction genes differentially expressed infloral parts and seeds are concerned with specifying flowers and seedsand their functions, and therefore modulations of such genes producevariant flowers and seeds.

Reproductive genes and gene products can function to either increase ordampen the phenotypes, biochemical activities and transcriptionprofiles, either in response to changes of internal plant programs or toexternal environmental fluctuations.

III.A.5.a. Use of Reproduction Genes, Gene Components and Products toModulate Phenotypes

The reproduction genes of the instant invention, when mutated oractivated differently, are capable of modulating one or more processesof flower, seed and fruit development. They are thus useful forimproving plants for agriculture and horticulture and for providingseeds with a better chemical composition for diverse industriesincluding the food, feed and chemical industries. Reproduction genes,gene components and products are useful to alter the following traitsand properties of plants, including development, such as flowering timeand number of inflorescences, flower development (including anther,stamen, pollen, style, stigma, ovary, ovule, and gametes), pollinationand fertilization (including sporogenesis gametogenesis, zygoteformation, embryo development, endosperm development, and malesterility, hybrid breeding systems and heterosis); cellular properties,such as cell size, cell shape, cell death, cell division, cellelongation, cell differentiation, and meiosis; organ characteristics,such as flowers, receptacle, sepals, petals, and tepals color, shape,and size, number, and petal drop); androecium, such as stamen (includinganther size, pollen sterile, size, shape, weight and color, number, andfilament size), gynoecium, such as carpel, ovary. number. and length)and style (stigma, ovule, size, shape, and number); pedicel and peduncle(size and shape), seeds, such as placenta, embryo. cotyledon, endosperm,suspensor, seed coat (testa), aleurone, development, including apomixis(gametophytic, apospory, diplospory), dormancy and germination; fruits,such as pericarp—thickness, texture (exocarp, mesocarp, endocarp);development (seed set, fruit set, false fruit, fruit elongation andmaturation, and dehiscence), and fruit drop; plant seed yield, such asincreased biomass, better harvest index, attraction of favorableinsects, better seed quality, and better yield of constituent chemicals;and plant population features, such as architecture (shade avoidance andplanting density).

To regulate any of the phenotype(s) above, activities of one or more ofthe reproduction genes or gene products can be modulated in an organismand tested by screening for the desired trait. Specifically, the gene,mRNA levels or protein levels can be altered in a plant using theprocedures described herein and the phenotypes can be assayed. As anexample, a plant can be transformed according to Bechtold and Pelletier(Methods Mol. Biol. 82:259-266 (1998)) and/or screened for variants asin Winkler et al. (Plant Physiol 118:743-50 (1998)) and visuallyinspected for the desired phenotype or metabolically and/or functionallyassayed.

III.A.5.b. Use of Reproduction Genes to Modulate Biochemical Activities

The activities of one or more of the reproduction genes can be modulatedto change biochemical or metabolic activities and/or pathways such asthose examples noted below. Such biological activities can be measuredaccording to the citations included in the Table below:

EXAMPLES OF BIOCHEMICAL/MOLECULAR FUNCTION/PROCESS ACTIVITIES ReferenceAND ASSAY Metabolism Energy production and Ap Rees, T. (1974). Inconversion “Plant Biochemistry. Glucosyl-transferase Biochemistry,Series One”, (CLONE_ID 1040) Vol. 11. (H. L. Kornberg and Heme-bindingprotein D. C. Phillips, eds.), (putative cytochrom Butterworths, London.B5) Juliano, B. O. and Varner, J. E. (CLONE_ID 3743) (1969). PlantPhysiol. Storage protein synthesis 44, 886-892. Inorganic ion transportand Bewley et al. (1993). Plant metabolism Physiol. Biochem. 31,483-490. Peroxidase Hills, M. J. and Beevers, H. (CLONE_ID 100990)(1987). Plant Physiol. 84, Cystathione beta 272-276. synthase Olsen, L.J. and Harada, J. J. (CLONE_ID 21847) (1991). In “Molecular Amino acidtransport and Approaches to metabolism Compartmentalization andl-asparaginase Metabolic Regulation (A. H. C. Huang (CLONE_ID 92780) andL. Taiz, eds.), Putative peptide/amino ASPP, Rockville, Md. acidMitsuhashi, W. and Oaks, A. transporter (1994). Plant Physiol. (CLONE_ID113723) 104, 401-407. Carbohydrate transport and Walker-Smith, D. J.,and metabolism Payne, J. W. (1985). Planta Glucose transport 164,550-556. protein Salmenkallio, M. and (CLONE_ID 33727) Sopanen, T.(1989). Plant Putative sugar Physiol. 89, 1285-1291. transporterBaumgartner, B. and (CLONE_ID 3250) Chrispeels, M. J. (1976). Starchbiosynthesis Plant Physiol. 58, 1-6. Coenzyme metabolism Elpidina, E. N.et al. (1991). Tyrosine Planta 185, 46-52. aminotransferase Ericson, M.C. and (ROOTY/SUPERROOT1) Chrispeels, M. J. (1973). (CLONE_ID 14570)Plant Physiol. 52, 98-104. Formate dehydrogenase Kern, R. andChrispeels, M. J. (CLONE_ID 7530) 1978) Plant Physiol. 62, Lipidmetabolism 815-819. Branched chain α- Dilworth, M. F. and Dure, L.ketoacid III. (1978). Plant Physiol. dehydrogenase E2 61, 698-702.subunit Chrispeels, M. J. and Jones, R. L. (CLONE_ID 25116) (1980/81).Isr. J. Bot. Acyl carrier protein-1 29, 222-245. (CLONE_ID 14291) Gould,S. E. B., and Rees, D. A. Lipid metabolic enzymes (1964). J. Sci. FoodSecretion Agric. 16, 702-709. Sensor protein RcsC- like (CLONE_ID 16461)Signal recognition particle RP54 (CLONE_ID 22158) Modulate floral organTranscriptional control Elliot et al. (1996). Plant number ANT(AP2-domain) DNA Cell 8, 155-168. binding protein Sakai et al. (2000).Plant SUP (Zinc finger) Cell 12, 1607-1618. Jacobsen and Meyerowitz(1997). Science 277, 1100-1103. Floral organ size Transcriptionalcontrol Mizukami et al. (2000). ANT (AP2-domain) DNA PNAS 97, 942-947.binding protein Krizek (1999). Developmental Genetics 25, 224-236.Female organ Membrane receptor kinase Clark and Meyerowitz number/Floralmeristem signal transduction (1997). Cell 89, 575-585 size CLV1 (LRRdomain and Jeong et al. (1999). Plant kinase domain) receptor Cell 11,1925-1934. CLV2 (LRR domain) Fletcher et al. (1999). receptor CLV3(Receptor ligand) Science 283, 1911-1914. Female reproduction DNAbinding protein Yanofsky et al. (1990). AG (MADS domain) DNA Nature 346,35-39. binding protein Female reproduction Signal transduction Kieber etal. (1993). Cell CTR1 (Raf kinase) 72, 427-441. Male organ number DNAmethylation Jacobsen and Meyerowitz MET1 (DNA (1997). Science 277,1100-1103. methyltransferase) Seed size control DNA binding proteinJofuku et al. (1994). Plant AP2 (AP2 domain) Cell 6, 1211-1225. RAP2(AP2 domain) US Patent #6,093,874; #5,994,622 Seed size control Polycombgroup protein Luo et al. (2000). PNAS 97, complex 10637-10642. FIE,FIS2, MEA Seed size control DNA methylation Scott et al. (2000). MET1Development 127, 2493-2502. Vinkenoog et al. (2000). Plant Cell 12,2271-2282. Luo et al. (2000). PNAS 97, 10637-10642. Embryo CAAT boxbinding complex Lotan et al. (1998). Cell 93, development/EmbryoLEC1/HAP3 1195. viability HAP2, HAP5 US Patent #6,235,975 Embryodevelopment/Seed DNA binding proteins Finkelstein et al. (1998).dormancy ABI4 (AP2 domain) Plant Cell 10, 1043-1054. FUS3 (B3 domain)Luerssen et al. (1998). Plant VP1 (B3 domain) J. 15, 755-764. Embryodevelopment Signal transduction Leung et al. (1994). Science ABI1, ABI2264, 1448-1452. [Serine/threonine protein Leung et al. (1997). Plantphosphatase 2C (PP2C)] Cell 9, 759-771. Endosperm development Chromatinlevel control of Ohad et al. (1996). PNAS gene activity 93, 5319-5324.Polycomb complex; FIE, US Patent #6,229,064 MEA, FIS2 Kiyosue et al.(1999). PNAS 96, 4186-4191. Grossniklaus et al. (1998). Science 280,446-450. Chaudhury et al. (1997) PNAS 94, 4223-4228. Integument DNAbinding Jofuku et al. (1994). Plant development/Seed coat AP2, ANT (AP2domain) Cell 6, 1211-1225. development Klucher et al. Plant Cell 8,137-153. BEL1 (Homeodomain) Reiser et al. (1995). Cell 83, 735-742.Anthocyanin production Secondary transporter Debeaujon et al. (2001).TT12 (MATE; multidrug and Plant Cell 13, 853-872. toxic compoundextrusion) Anthocyanin production DNA binding protein Nesi et al.(2000). Plant Cell TT8 (Basic helix-loop-helix 12, 1863-1878. domain)Fruit development Chromatin level control of Ohad et al. (1996). PNASgene activity 93, 5319-5324. Polycomb complex; FIE, Kiyosue et al.(1999). MEA, FIS2 PNAS 96, 4186-4191. Grossniklaus et al. (1998).Science 280, 446-450. Chaudhury et al. (1997) PNAS 94, 4223-4228. Fruitsize control Signal transduction Frary et al. (2000). Science FW2.2(c-Ras P21) 289, 85-88. Fruit development/Pod Transcriptional controlLiljegren et al. (2000). shattering SHP1, SHP2, FUL (MADS Nature 404,766-770. domain) DNA binding Ferrandiz et al. (2000). proteins Science289, 436-438.. Transcription and Transcription Delseny, M. et al.(1977). Posttranscription SRF-domain AGL11 Planta 135, 125-128.(CLONE_ID 32791) Lalonde, L. and Bewley, J. D. AP2-domain containing(1986). J. Exp. Bot. 37, protein (CLONE_ID 754-764. 332) Walling, L. etal. (1986). Myb-DNA binding PNAS 83, 2123-2125. protein Okamuro, J. K.and (CLONE_ID 94597) Goldberg, R. B. (1989). In Transcription factors“Biochemistry of Plants, Signal transduction Vol 15.” Academic Press,mechanisms Inc. Protein-kinases Wong, J. et al. (1995). PhosphatasesGenes Dev. 9, 2696-2711. meiosis proteins Dimitrov et al. (1994). J.Chromatin remodeling Cell Biol. 126, 591-601. proteins Landsberger, N.and Chaperones Wolffe, A. P. (1997). Chalcone synthase EMBO J. 16,4361-4373. Putative Ser/Thr protein Bogdanove, A. J. and kinase(CLONE_ID Martin, G. G. (2000). PNAS 31383) 97, 8836-8840. ER6-likeprotein Zhu, H. et al. Science Jul. (implicated in ethylene 26, 2001:signal transduction) 10.1126/science.1062191 (CLONE_ID 7474) (Reports).Translation, ribosomal structure and biogenesis Ribosomal proTein S15A(CLONE_ID 17466) Translation initiation factor (CLONE_ID 103464)Posttranslational modification, protein turnover, chaperones DnaJ-domaincontaining protein (CLONE_ID 4150) Cyclophilin-like protein (CLONE_ID35643) Cell division and Repair Cell division and Rogan, P. G. andSimon, E. W. chromosome partitioning (1975). New Phytol. 74, Protein ofunknown 273-275. function Morahashi, Y. and Bewley, J. D. withtropomyosin-, (1980). Plant Physiol myosin 66, 70-73. tail- andfilament- Morahashi, Y. et al. (1981). domains Plant Physiol. 68,318-323. (CLONE_ID 15546) Morahashi, Y. (1986). Actin-1 Physiol. Plant.66, 653-658. (CLONE_ID 25785) Zlatanova, J. et al. (1987). DNAreplication, Plant Mol. Biol. 10, 139-144. recombination and repairZlatanova, J. and Ivanov, P. Proliferating cell (1988). Plant Sci. 58,71-76. nuclear antigen-1 (axillary protein, DNA polymerase I delta)(CLONE_ID 28554) AAA-type ATPase, cdc48 (CLONE_ID 100292) Cell envelopebiogenesis, outer membrane dTDP-D-glucose 4,6- dehydratase (CLONE_ID28597) Putative cinnamoyl- CoA reductase (CLONE_ID 109228)

Other biological activities that are modulated by the reproductive genesand gene products are listed in the Reference tables. Assays fordetecting such biological activities are described in the Protein Domaintable, for example.

III.5.A.c. Use of Reproduction Genes, Gene Components and Products toModulate Transcription Levels

Reproduction genes are characteristically differentially transcribed inresponse to cell signals such as fluctuating hormone levels orconcentrations, whether internal or external to an organism or cell.Many reproduction genes belong to networks or cascades of genes underthe control of regulatory genes. Thus some reproduction genes are usefulto modulate the expression of other genes. Examples of transcriptionprofiles of reproduction genes are described in the Table below withassociated biological activities. “Up-regulated” profiles are thosewhere the mRNA transcript levels are higher in flowers, flower parts orsiliques as compared to the plant as a whole. “Down-regulated” profilesrepresent higher transcript levels in the whole plant as compared toflowers, flower parts or siliques alone.

EXAMPLES OF BIOCHEMICAL PHYSIOLOGICAL ACTIVITIES OF TYPE OF GENESCONSEQUENCES GENES WITH TRANSCRIPT WITH ALTERED OF ALTERING ALTEREDLEVELS ACTIVITY GENE EXPRESSION EXPRESSION Up Regulated Genes thatcontrol Flowers form from Transcription Factors Transcripts flowerdifferentiation, flower meristem Signal transduction Flower number andsize Floral organs mature Membrane Structure Reproduction Genes thatpromote Flavonoid pathways Protein kinases Genes petal, stamen andinduced Phosphatases carpel formation Meiosis proteins Genes controllingChromatin flower-specific remodeling proteins metabolism such asChaperones petal pigments Chalcone synthase Genes that promote Aminoacid transport ovule formation and metabolism Genes that promote Storageprotein fertilization, seed, synthesis embryo and Lipid metabolicendosperm formation enzymes Carbohydrate transport and metabolism Starchbiosynthesis AP2 Genes activated by Many steps and Proteins associatedReproduction AP2 transcription pathways induced, with: Genes factorsdevelopmental and Energy production Genes that induce metabolic andconversion petal and stamen No petals or stamens Amino acid transportformation produced and metabolism Carbohydrate transport and metabolismLipid metabolism Transcription and signal transduction Poortranslational modification DNA replication Chromatin remodelingDown-Regulated Genes that repress Flowers form from Transcritipionfactors Transcripts flower development flower meristem Signaltransduction Flower pathways Reproduction Kinases and Genes phosphatasesGenes that induce Non-floral organs are Chromatin stem, leaf and otherrepressed remodeling proteins organ differentiation Genes thatnegatively Flower-specific regulate flower pathways are induced specificmetabolism Genes that negatively regulate ovule formation, meiosis,fertilization and seed development AP2 Reproduction Genes activated byMany steps and Proteins associated Genes AP2 transcription pathwaysinduced, with: factors developmental and Energy production metabolic andconversion Genes that induce No petals or stamens Amino acid transportpetal and stamen produced and metabolism formation Carbohydratetransport and metabolism Lipid metabolism Transcription and signaltransduction Poor translational modification DNA replication Chromatinremodeling

While polynucleotides and gene products modulating reproduction can actalone, combinations of these polynucleotides also affect growth anddevelopment. Useful combinations include different polynucleotidesand/or gene products of the instant invention that have similartranscription profiles or similar biological activities, and members ofthe same or similar biochemical pathways. In addition, the combinationof a polynucleotide and/or gene product(s) capable of modulatingreproduction with a hormone responsive polynucleotide, particularly oneaffected by gibberellic acid and/or Auxin, is also useful because of theinteractions that exist between hormone-regulated pathways, anddevelopment. Here, in addition to polynucleotides having similartranscription profiles and/or biological activities, useful combinationsinclude polynucleotides that may have different transcription profilesbut which participate in common or overlapping pathways.

Use of Promoters and Reproduction Genes

Promoter of reproduction genes are useful for transcription of desiredpolynucleotides, both plant and non-plant. For example, extra copies ofcarbohydrate transporter genes can be operably linked to a reproductiongene promoter and inserted into a plant to increase the “sink” strengthof flowers or siliques. Similarly, reproduction gene promoters can beused to drive transcription of metabolic enzymes capable of altering theoil, starch, protein or fiber of a flower or silique. Alternatively,reproduction gene promoters can direct expression of non-plant genesthat can, for instance confer insect resistance specifically to aflower.

III.A.7. Ovule Genes, Gene Components and Products

The ovule is the primary female sexual reproductive organ of floweringplants. It contains the egg cell and, after fertilization occurs,contains the developing seed. Consequently, the ovule is at timescomprised of haploid, diploid and triploid tissue. As such, ovuledevelopment requires the orchestrated transcription of numerouspolynucleotides, some of which are ubiquitous, others that areovule-specific and still others that are expressed only in the haploid,diploid or triploid cells of the ovule.

Although the morphology of the ovule is well known, little is known ofthese polynucleotides and polynucleotide products. Mutants allowidentification of genes that participate in ovule development. As anexample, the pistillata (PI) mutant replaces stamens with carpels,thereby increasing the number of ovules present in the flower.Accordingly, comparison of transcription levels between the wild-typeand PI mutants allows identification of ovule-specific developmentalpolynucleotides.

Changes in the concentration of ovule-specific polynucleotides duringdevelopment results in the modulation of many polynucleotides andpolynucleotide products. Examples of such ovule-specific responsivepolynucleotides and polynucleotide products are shown in the Reference,Sequence, Protein Group, Protein Group Matrix, MA_diff, and MA_clusttables. These polynucleotides and/or products are responsible foreffects on traits such as fruit production and seed yield.

While ovule-specific developmentally responsive polynucleotides andpolynucleotide products can act alone, combinations of thesepolynucleotides also affect fruit and seed growth and development.Useful combinations include different ovule-specific developmentallyresponsive polynucleotides and/or polynucleotide products that havesimilar transcription profiles or similar biological activities, andmembers of the same or similar biochemical pathways. In addition, thecombination of an ovule-specific developmentally responsivepolynucleotide and/or polynucleotide product with an environmentallyresponsive polynucleotide is also useful because of the interactionsthat exist between development, hormone-regulated pathways, stresspathways and nutritional pathways. Here, in addition to polynucleotideshaving similar transcription profiles and/or biological activities,useful combinations include polynucleotides that may have differenttranscription profiles but which participate in a common pathway. TheMA_diff Table(s) reports the transcript levels of the experiment (seeEXPT ID: 108595). For transcripts that had higher levels in the samplesthan the control, a “+” is shown. A “−” is shown for when transcriptlevels were reduced in root tips as compared to the control. For moreexperimental detail see the Example section below.

Ovule genes are those sequences that showed differential expression ascompared to controls, namely those sequences identified in the MA_difftables with a “+” or “−” indication.

Ovule Genes Identified by Cluster Analyses of Differential Expression

Ovule Genes Identified by Correlation to Genes that are DifferentiallyExpressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of Ovule genes is any group in the MA_dust thatcomprises a cDNA ID that also appears in Expt ID 108595 of the MA_difftable(s).

Ovule Genes Identified by Correlation to Genes that Cause PhysiologicalConsequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of Ovulegenes. A group in the MA_clust is considered a Ovule pathway or networkif the group comprises a cDNA ID that also appears in Knock-in orKnock-out tables that causes one or more of the phenotypes described insection above.

Ovule Genes Identified by Amino Acid Sequence Similarity

Ovule genes from other plant species typically encode polypeptides thatshare amino acid similarity to the sequences encoded by corn andArabidopsis Ovule genes. Groups of Ovule genes are identified in theProtein Group table. In this table, any protein group that comprises apeptide ID that corresponds to a cDNA ID member of a Ovule pathway ornetwork is a group of proteins that also exhibits Ovulefunctions/utilities.

Such ovule-specific developmentally responsive polynucleotides andpolynucleotide products can function to either increase or dampen theabove phenotypes or activities either in response to transcript changesduring ovule development or in the absence of ovule-specificpolynucleotide fluctuations. More specifically, ovule-specificdevelopmentally responsive polynucleotides and polynucleotide productsare useful to or modulate one or more of the phenotypes, including eggcell, maturation (for development of parthenogenic embryos), metabolism,polar nuclei, fusion (for development of parthenogenic endosperm),central cell, maturation, metabolism (for alteration of endospermmetabolism), synergids, maturation, programmed cell death, nucellus,maturation, integuments, maturation, funiculus, extension (for increasedseed), cuticle, maturation, tensile properties (for increased seedsize), ovule, modulation of ovule senescence, and shaping (for increasedseed number).

To produce the desired phenotype(s) above, one or more of theovule-specific developmentally responsive polynucleotides andpolynucleotide products can be tested by screening for the desiredtrait. Specifically, the polynucleotide, mRNA levels, or protein levelscan be altered in a plant utilizing the procedures described herein andthe phenotypes can be assayed. As an example, a plant can be transformedaccording to Bechtold and Pelletier (1998, Methods. Mol. Biol.82:259-266) and visually inspected for the desired phenotype ormetabolically and/or functionally assayed according to Weigel et al.(2000, Plant Physiol 122: 1003-14) and Winkler et al. (1998, PlantPhysiol 118: 743-50).

Alternatively, the activities of one or more of the ovule-specificdevelopmentally responsive polynucleotides and polynucleotide productscan be modulated to change biochemical or metabolic activities and/orpathways such as those noted below. Such biological activities can bemeasured according to the citations included in the Table below:

BIOCHEMICAL OR GENERAL METABOLIC ACTIVITIES CATEGORY AND/OR PATHWAYSASSAY Cell Growth and Programmed Cell Death Pennell and LambDifferentiation (1997) Plant Cell 9, 1157-1168 DNA Methylation and Adamset al. (2000) Imprinting Development 127: 2493-502 Organ Growth andOvule Growth and De Martinis and Development Development Mariani (1999)Ethylene Response Plant Cell 11: Megagametophyte 1061-72 DevelopmentChristensen et al. Seed Growth and (1997) Sexual Plant DevelopmentReproduc 10: 49-64 Fertilization Scott et al. (1998) IndependentDevelopment 125: Seed Development 3329-41 Ohad et al. (1996) PNAS USA93: 5319-24 Chaudhury et al. (1997) PNAS USA 94: 4223-28 SignalTransduction Ethylene Metabolism DeMartinis and Protein RemodelingMariani (1999) Sucrose Mobilization Plant Cell 11: and 1061-1072Partitioning Winkler et al. Pollen Tube Adhesion (1998) Plant JasmonicAcid Physiol 118: Biosynthesis 743-750 Senescence and Cell ApomixisDeath Environmental Wound and Defense Epple and Responses Response GeneBohlmann (1997) Expression Plant Cell 9: 509-20 Stress Response He etal. (1998) Plant J. 14: 55-63

Other biological activities that can be modulated by the ovule-specificdevelopmentally responsive polynucleotides and polynucleotide productsare listed in the Reference tables. Assays for detecting such biologicalactivities are described in the Protein Domain table section.

Ovule-specific developmentally responsive polynucleotides arecharacteristically differentially transcribed in response to fluctuatingdevelopmental-specific polynucleotide levels or concentrations, whetherinternal or external to a cell. The MA_diff Table reports the changes intranscript levels of various ovule-specific developmentally responsivepolynucleotides in ovules.

These data can be used to identify a number of types of ovule-specificdevelopmentally responsive polynucleotides. Profiles of these differentovule-specific developmentally responsive polynucleotides are shown inthe Table below with examples of associated biological activities.

EXAMPLES OF TRANSCRIPTS PHYSIOLOGICAL BIOCHEMICAL AFFECTED BY TYPES OFGENES CONSEQUENCES ACTIVITY Ethylene Signals Responders to EthylenePerception Transcription Ethylene Ethylene Uptake Factors Modulation ofEthylene Transporters Response Transduction Pathways Specific GeneTranscription Initiation Protein Repression of Pathways InhibitTransport of Remodeling to Optimize Abscissic Abscissic acid acidResponse Pathways Degradation Lower at 1 hours High Abscissic acidAbscissic acid than 6 hours Responders Metabolic Pathways Repressor ofAbscissic Negative Regulation of acid Deprivation Abscissic acidPathways Pathways

Use of Promoters of Ovule Genes

Promoters of Ovule genes are useful for transcription of any desiredpolynucleotide or plant or non-plant origin. Further, any desiredsequence can be transcribed in a similar temporal, tissue, orenvironmentally specific patterns as the Ovule genes where the desiredsequence is operably linked to a promoter of a Ovule gene. The proteinproduct of such a polynucleotide is usually synthesized in the samecells, in response to the same stimuli as the protein product of thegene from which the promoter was derived. Such promoter are also usefulto produce antisense mRNAs to down-regulate the product of proteins, orto produce sense mRNAs to down-regulate mRNAs via sense suppression.

III.A.8. Seed and Fruit Development Genes, Gene Components and Products

The ovule is the primary female sexual reproductive organ of floweringplants. At maturity it contains the egg cell and one large central cellcontaining two polar nuclei encased by two integuments that, afterfertilization, develops into the embryo, endosperm, and seed coat of themature seed, respectively. As the ovule develops into the seed, theovary matures into the fruit or silique. As such, seed and fruitdevelopment requires the orchestrated transcription of numerouspolynucleotides, some of which are ubiquitous, others that areembryo-specific and still others that are expressed only in theendosperm, seed coat, or fruit. Such genes are termed fruit developmentresponsive genes.

Changes in the concentration of fruit-development responsivepolynucleotides during development results in the modulation of manypolynucleotides and polynucleotide products. Examples of such fruitdevelopment responsive polynucleotides and polynucleotide productsrelative to leaves and floral stem are shown in the Reference, Sequence,Protein Group, Protein Group Matrix, MA_diff, MA_clust, Knock-in andKnock-out tables. The polynucleotides were discovered by isolatingfruits at developmental stages from Arabidopsis wild-type ecotype“Wassilewskija”, and measuring the mRNAs expressed in them relative tothose in a leaf and floral stem sample. These polynucleotides and/orproducts are responsible for effects on traits such as seed size, seedyield, seed composition, seed dormancy, fruit ripening, fruitproduction, and pod shattering.

While fruit development responsive polynucleotides and polynucleotideproducts can act alone, combinations of these polynucleotides alsoaffect fruit and seed growth and development. Useful combinationsinclude different polynucleotides and/or polynucleotide products thathave similar transcription profiles or similar biological activities,and members of the same or functionally similar biochemical pathways. Inparticular, modulation of transcription factors and/or signaltransduction pathways are likely to be useful for manipulating wholepathways and hence phenotypes. In addition, the combination ofovule-developmentally responsive polynucleotides and/or polynucleotideproducts with environmentally responsive polynucleotides is also usefulbecause of the interactions that exist between development,hormone-regulated pathways, stress and pathogen induced pathways andnutritional pathways. Here, useful combinations include polynucleotidesthat may have different transcription profiles, and participate incommon or overlapping pathways but combine to produce a specific,phenotypic change.

Such fruit development responsive polynucleotides and polynucleotideproducts can function to either increase or dampen the above phenotypesor activities either in response to transcript changes in fruitdevelopment or in the absence of fruit development polynucleotidefluctuations.

The MA_diff Table(s) reports the transcript levels of the experiment(see EXPT ID: 108436, 108437, 108438). For transcripts that had higherlevels in the samples than the control, a “+” is shown. A “−” is shownfor when transcript levels were reduced in root tips as compared to thecontrol. For more experimental detail see the Example section below.

Fruit genes are those sequences that showed differential expression ascompared to controls, namely those sequences identified in the MA_difftables with a “+” or “−” indication.

Fruit Genes Identified by Cluster Analyses of Differential Expression

Fruit Genes Identified by Correlation to Genes that are DifferentiallyExpressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of Fruit genes is any group in the MA_clust thatcomprises a cDNA ID that also appears in Expt ID 108436, 108437, 108438of the MA_diff table(s).

Fruit Genes Identified by Correlation to Genes that Cause PhysiologicalConsequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of Fruitgenes. A group in the MA_clust is considered a Fruit pathway or networkif the group comprises a cDNA ID that also appears in Knock-in orKnock-out tables that causes one or more of the phenotypes described insection above.

Fruit Genes Identified by Amino Acid Sequence Similarity

Fruit genes from other plant species typically encode polypeptides thatshare amino acid similarity to the sequences encoded by corn andArabidopsis Fruit genes. Groups of Fruit genes are identified in theProtein Group table. In this table, any protein group that comprises apeptide ID that corresponds to a cDNA ID member of a Fruit pathway ornetwork is a group of proteins that also exhibits Fruitfunctions/utilities.

Use of Fruit Development Responsive Genes to Modulate Phenotypes

Manipulation of the polynucleotides in the mature ovule, developingembryo, endosperm, seed coat and fruit enables many features of seed andfruit to be improved including the following:

-   -   Female fertility, megasporogenesis, embryo and endosperm        development, ovule size, endosperm size, embryo size, seed size,        seed yield, seed protein, seed oil, seed starch, seed cell        number, cell size, seed coat development, organ size, dormancy        and acquisition of desiccation tolerance, seed storage and        longevity, seed germination, apomixis, production of seedless        fruit and vegetables and hybrid seed production.

To improve any of the phenotype(s) above, activities of one or more ofthe fruit development responsive polynucleotides and polynucleotideproducts can be modulated and the plants can be tested by screening forthe desired trait. Specifically, the polynucleotide, mRNA levels, orprotein levels can be altered in a plant utilizing the proceduresdescribed herein and the phenotypes can be assayed. As an example, aplant can be transformed according to Bechtold and Pelletier (1998,Methods. Mol. Biol. 82:259-266) and visually inspected for the desiredphenotype or metabolically and/or functionally assayed.

Use of Fruit Development Responsive Genes to Modulate BiochemicalActivities

The activities of one or more of the fruit-expressed polynucleotides andpolynucleotide products can be modulated to change biochemical ormetabolic activities and/or pathways such as those noted below. Suchbiological changes can be achieved and measured according to citationssuch as the following:

-   1. Winkler et al. (1998). Plant Physiol. 118, 743-750-   2. Weigel et al. (2000). Plant Physiol. 122, 1003-1014-   3. Cosgrove (1997). Plant Cell 9, 1031-1041-   4. Jacobs (1997). Plant Cell 9, 1021-1029-   5. Reismeier et al. (1994). EMBO J. 13, 1-7-   6. Carland et al. (1999). Plant Cell 11, 2123-2138-   7. Cheng et al. (1996). Plant Cell 8, 971-983-   8. Weber et al. (1995). Plant Cell 7, 1835-1846-   9. Leyser and Furner (1992). Development 116, 397-403-   10. Hayashi et al. (1998). Plant Cell 10, 183-196.-   11. Pyke (1999). Plant Cell 11, 549-556-   12. Lotan et al. (1998). Cell 93, 1195-1205-   13. Lending and Larkins (1989). Plant Cell 1, 1011-1023-   14. Hong et al. (1996). Development 122, 2051-2058.-   15. Fernandez et al. (2000). Science 289, 436-438-   16. D'Aoust et al. (1999). Plant Cell 11, 2407-2418-   17. Bewley (1997). Plant Cell 9, 1055-1066-   18. Heath et al. (1986). Planta 169, 304-312-   19. Browse et al. (1986). Anal. Biochem. 152, 141-145-   20. D'Aoust et al. (1999). Plant Cell 11, 2407-2418

Other biological activities that can be modulated by the fruit-specificdevelopmentally responsive polynucleotides and polynucleotide productsare listed in Reference Tables. Assays for detecting such biologicalactivities are described in the table as well as in the Protein Domaintables.

BIOLOGICAL FUNCTION UTILITY CITATION ASSAY CITATION Ovule Growth,Ethylene and Manipulate De Martinis Analyze Winkler et al. Ovuleethylene female and Mariani ovule and (1998). Plant Development signalfertility. (1999). Plant seed Physiol. 118, and Seed transductionManipulate Cell 11, 1061-1072. development 743-750. Growth and pathwaymegasporo- Silencing by light Systematic Development Examples: genesis.gene microscopy reverse AP2 domain Manipulate expression of or bygenetics of DNA binding female the ethylene- confocal transfer-proteins; gametophyte forming microscopy. DNA-tagged EREBP, EBFdevelopment. enzyme results Test for lines of Example: Manipulate in areversible fertilization Arabidopsis. Leucine-rich fertilizationinhibition of independent Weigel et al. receptor independent ovuleendosperm (2000). Plant kinase; ETR- endosperm development indevelopment. Physiol 122, like development. transgenic Test for1003-1014. Example: Raf Manipulate tobacco plants. fertilizationActivation kinase; CTR fertilization Christensen et independent taggingin independent al. (1997). embryo Arabidopsis. embryo Sexual Plantdevelopment. Ohad et al. development. Reproduc. 10, Test for (1996).PNAS Manipulate 49-64. fertilization USA 93, fertilizationMegagametogenesis independent 5319-5324. A independent in seed mutationthat seed Arabidopsis production. allows development. wild type andAnalyze seed endosperm Manipulate the Gf mutant. size. development ovulesize. Christiansen Analyze seed without Manipulate and Drews, yield.fertilization endosperm unpublished Analyze seed Chaudhury et size.composition. al. (1997). Manipulate Analyze fruit PNAS USA embryo size.size. 94, 4223-4228. Manipulate Fertilization- seed size. independentManipulate seed seed yield. development Manipulate in Arabidopsis seedprotein. thaliana Manipulate De Martinis seed oil. and MarianiManipulate (1999). Plant starch Cell 11, 1061-1072. production.Silencing Manipulate gene cell number. expression of Manipulate theethylene- cell size. forming Produce enzyme results seedless fruit in areversible and inhibition of vegetables ovule Manipulate development infruit size. transgenic tobacco plants. Christensen et al. (1997). SexualPlant Reproduc. 10, 49-64. Megagametogenesis in Arabidopsis wild typeand the Gf mutant. Scott et al. (1998). Development 125, 3329-3341.Parent- of-origin effects on seed development in Arabidopsis thalianaHeath et al. (1986). Planta 169, 304-312. Browse et al. (1986). Anal.Biochem. 152, 141-145. D'Aoust et al. (1999). Plant Cell 11, 2407-2418.2. Growth Manipulate Wilson et al. Analyze ovule Winkler et al. andfemale (1996). Plant and seed (1998). Plant developmental fertility.Cell 8, 659-671. A development Physiol. 118, control Manipulatedissociation by light 743-750. genes megasporo- insertion microscopy orSystematic — genesis. causes a by confocal reverse UpregulatedManipulate semidominant microscopy. genetics of genes female mutationthat Test for transfer- Example: gametophyte increases fertilizationDNA-tagged DNA binding development. expression of independent lines ofproteins; tiny- Manipulate TINY, an endosperm Arabidopsis. like, AGL1,fertilization Arabidopsis development. Weigel et al. FBP2, AGL9,independent gene related to Test for (2000). Plant AP3, CPC- endospermAPETALA2. fertilization Physiol 122, like myb. development. Zhao et alindependent 1003-1014. Example: Manipulate (1999). embryo ActivationProtein fertilization Developmental development. tagging in kinase;independent Genetics 25, Test for Arabidopsis. ASK1. embryo 209-223. Thefertilization Ohad et al. Example: development. ASK1 gene independent(1996). PNAS Auxin Manipulate regulates seed USA 93, conjugatingfertilization development production. 5319-5324. A enzyme; independentand interacts Analyze seed mutation that indole-3- seed with the UFOsize. allows acetate beta- development. gene to Analyze seed endospermglucosyltransferase. Manipulate control floral yield. developmentExample: S/T ovule size. organ identity Analyze seed without proteinManipulate in composition. fertilization kinase; endosperm Arabidopsis.Analyze fruit Chaudhury et APK1. size. Flanagan et size. al. (1997).Example: Manipulate al. (1996). Analyze PNAS USA Leucine-rich embryosize. Plant J. 10, seedling size. 94, 4223-4228. receptor Manipulate343-53. Analyze Fertilization- kinase; organ size Specific seedlingindependent CLV1, ER, and number. expression of viability. seed BRI,Cf-2- Manipulate the AGL1 Screen for development like. seed size.MADS-box changes in in — Manipulate gene suggests shatter time.Arabidopsis Downregulated seed yield. regulatory Screen for thalianagenes Manipulate functions in changes in De Martinis Example: seedlingsize Arabidopsis germination and Mariani Cyclin- through seed gynoeciumfrequency. (1999). Plant dependent size. and ovule Screen for Cell 11,1061-1072. kinase; cdc2. Manipulate development. seed longevitySilencing seedling vigor Angenent et and viability. gene through seedal. (1994). expression of size. Plant J 1994. the ethylene- Manipulate5, 33-44. Co- forming seed protein. suppression of enzyme resultsManipulate the petunia in a reversible seed oil. homeotic geneinhibition of Manipulate fbp2 affects ovule starch the identity ofdevelopment in production. the generative transgenic Manipulatemeristem. tobacco plants. integument AGL9 web Christensen etdevelopment. page. al. (1997). Manipulate Wada et al. Sexual Plantseedcoat (1997) Reproduc. 10, development. Science 277, 49-64.Manipulate 1113-6. Megagametogenesis cell size. Epidermal cell inManipulate differentiation Arabidopsis cell number. in Arabidopsis wildtype and Manipulate determined by the Gf mutant. homeotic a Myb Scott etal. gene homolog (1998). expression. CPC. Development ManipulateSzerszen et al. 125, 3329-3341. organ size. (1994). Parent- ManipulateScience 16, of-origin meristem size. 1699-1701. effects on Produceiaglu, a gene seed seedless fruit from Zea development and mays invegetables involved in Arabidopsis Manipulate conjugation of thaliana.fruit size. growth Heath et al. Manipulate hormone (1986). time of seedindole-3- Planta 169, dispersal. acetic acid. 304-312. Manipulate Ito etal. Browse et al. seed viability (1997). Plant (1986). Anal. uponstorage. Cell Physiol. Biochem. Manipulate 38, 248-258. A 152, 141-145.germination serine/threonine D'Aoust et al. frequency. protein (1999).Plant kinase gene Cell 11, 2407-2418. isolated by an in vivo bindingprocedure using the Arabidopsis floral homeotic gene product, AGAMOUS.Clark et al. (1997). Cell 89, 575-585. The CLAVATA1 gene encodes aputative receptor kinase that controls shoot and floral meristem size inArabidopsis. Torii et al. (1996). Plant Cell 8, 735-746. The ArabidopsisERECTA gene encodes a putative receptor protein kinase withextracellular leucine-rich repeats. Li and Chory (1997). Cell 90,929-38. A putative leucine-rich repeat receptor kinase involved inbrassinosteroid signal transduction. 3. Cell Manipulate Solomon et al.Analyze ovule Winkler et al. senescence female (1999). Plant and seed(1998). Plant and cell death fertility. Cell 11, 431-444. developmentPhysiol. 118, Example: Manipulate The by light 743-750. Cystatin seedset. involvement of microscopy or Systematic Example: Manipulatecysteine by confocal reverse WIPK seed yield. proteases and microscopy.genetics of Manipulate protease Analyze seed transfer- seed size.inhibitor genes set. DNA-tagged Manipulate in the Analyze seed lines offruit set. regulation of size. Arabidopsis. Promote programmed Analyzeseed Weigel et al. apomixis. cell death in yield. (2000). Plant Produceplants. Analyze fruit Physiol 122, Seedless fruit Zhang et al. set.1003-1014. and (2000). Plant J. Screen for Activation vegetables. 23,339-347. fertilization tagging in Multiple levels independentArabidopsis. of tobacco seed Ohad et al. WIPK development. (1996). PNASactivation USA 93, during the 5319-5324. A induction of cell mutationthat death by fungal allows elicitins. endosperm development withoutfertilization 4. Protein Manipulate Christensen et Test for Winkler etal. remodeling female al. (1997). altered female (1998). Plant Example:fertility. Sexual Plant fertility, seed Physiol. 118, DNA-J ManipulateReproduc. 10, set, seed 743-750. protein/chaperones female 49-64. yield.Systematic gametophyte Megagametogenesis Analyze ovule reversedevelopment. in development genetics of Promote Arabidopsis by lighttransfer- apomixis. wild type and microscopy or DNA-tagged Manipulatethe Gf mutant. by confocal lines of endosperm Cory microscopy.Arabidopsis. development. Christiansen Analyze seed Weigel et al.Manipulate and Gary size. (2000). Plant embryo Drews, Analyze seedPhysiol 122, development. unpublished yield. 1003-1014. ManipulateAnalyze seed Activation seed size. composition. tagging in ManipulateArabidopsis. seed yield. Christensen et Manipulate al. (1997). seedprotein. Sexual Plant Manipulate Reproduc. 10, seed oil. 49-64.Manipulate Megagametogenesis starch. in Produce Arabidopsis seedlessfruit wild type and and the Gf mutant. vegetables. Ohad et al. (1996).PNAS USA 93, 5319-5324. A mutation that allows endosperm developmentwithout fertilization Scott et al. (1998). Development 125, 3329-3341.Parent- of-origin effects on seed development in Arabidopsis thaliana.Heath et al. (1986). Planta 169, 304-312. Browse et al. (1986). Anal.Biochem. 152, 141-145. D'Aoust et al. (1999). Plant Cell 11, 2407-2418.5. Sucrose Manipulate Mapping of Analyze ovule Winkler et al.mobilization and female tomato genes and seed (1998). Plant partitioningfertility. associated development Physiol. 118, Example: Manipulate withsugar by light 743-750. Invertase ovule metabolism. microscopy orSystematic inhibitor development. Tomato by confocal reverse Example:Manipulate Genetics Co- microscopy. genetics of bZIP seed op Report 48,Determine transfer-DNA- transcription development. 22-23 (1998) femaletagged lines of factor Manipulate Ikeda et al. fertility. Arabidopsis.(translation of endosperm (1999). Plant Analyze seed Weigel et al. bZIPprotein development. Physiol 121, mass. (2000). Plant is inhibited byManipulate 813-820. Analyze seed Physiol 122, sucrose levels embryoSucrose and yield. 1003-1014. greater than development. CytokininAnalyze seed Activation 25 mM) Manipulate Modulation of composition.tagging in Example: seed size. WPK4, a Analyze Arabidopsis. LipoxygenaseManipulate Gene organ size. Christensen et — seed yield. Encoding aAnalyze al. (1997). Downregulated Manipulate SNF1-Related seedling size.Sexual Plant gene seed protein. Protein Analyze Reproduc. 10, Example:Manipulate Kinase from seedling 49-64. SNF1-related seed oil. Wheat.viability. Megagametogenesis protein kinase Manipulate Rook et al. instarch. (1998). Plant Arabidopsis Manipulate J. 15, 253-263. wild typeand cell size. Sucrose- the Gf mutant. Manipulate specific Ohad et al.cell number. signaling (1996). PNAS Manipulate represses USA 93, organsize. translation of 5319-5324. A Manipulate the mutation that meristemArabidopsis allows size. ATB2 bZIP endosperm Manipulate transcriptiondevelopment seedling size factor gene. without through seed Rook et al.fertilization size. (1998). Plant Scott et al. Manipulate Mol Biol(1998). seedling 37, 171-178. Development viability The light- 125,3329-3341. through seed regulated Parent- size. Arabidopsis of-originProduce bZIP effects on seedless fruit transcription seed and factorgene development vegetables. ATB2 in Translational encodes a Arabidopsiscontrol of protein with thaliana. gene an unusually 6. Heath et al.expression in long leucine (1986). ovule and zipper Planta 169, seed bydomain. 304-312. sucrose. Bunker et al. 7. Browse et Manipulate (1995).Plant al. (1986). assimilate Cell 7, 1319-1331. Anal. partitioning inSink Biochem. ovule and limitation 152, 141-145. seed induces the 8.D'Aoust et development. expression of al. (1999). multiple Plant Cell11, soybean 2407-2418. vegetative lipoxygenase mRNAs while theendogenous jasmonic acid level remains low. Lowry et al. (1998). PlantPhysiol. 116, 923-933. Specific soybean lipoxygenases localize todiscrete subcellular compartments and their mRNAs are differentiallyregulated by source-sink status. 6. Jasmonic Targeted Sanders et al.Test for Winkler et al. acid death of cells (2000). Plant altered female(1998). Plant biosynthesis belonging to Cell 12, 1041-1062. fertility.Physiol. 118, and the female The Analyze male 743-750. signalgametophyte, Arabidopsis fertility. Systematic transduction ovule orDELAYED Screen for reverse pathway integuments. DEHISCENCE1 enhancedgenetics of Example: Delay gene expression of transfer- Biosyntheticsenescence of encodes an pathogen DNA-tagged enzyme; unfertilized enzymein the defense lines of FMN female jasmonic acid response Arabidopsisoxidoreductase gametophyte, synthesis genes. Weigel et al. 12- ovule orpathway. (2000). Plant oxophyto- integuments. Vijayan et al. Physiol122, dienoate Manipulate (1998). A role 1003-1014. reductase, female forjasmonate Activation OPR1, OPR1- fertility in pathogen tagging in like.Coordinate defense of Arabidopsis. Example: female with Arabidopsis.Signal male PNAS USA transduction reproduction. 95, 7209-7214. pathwayManipulate Seo et al. kinase WIPK. male fertility. (1999). PlantEnhanced Cell 11, 289-298. defense Jasmonate- response in based woundovules and signal seed transduction requires activation of WIPK, atobacco mitogen- activated protein kinase. Environmental 1. Wound andPathogen Song et al. Resistance to Winkler et al. responses defenseresistant (1995). Xanthamonas (1998). Plant response gene ovules.Science 270, sp. Physiol. 118, expression Pathogen 1804-1806. AResistance to 743-750. Example: resistant receptor known SystematicLeucine rich seeds. kinase-like arabidopsis reverse receptor S/TPathogen protein pathogens in genetics of kinase; Xa21- resistant fruit.encoded by the ovules, seed transfer-DNA- like and rice disease andfruit. tagged lines of TMK-like. resistance Arabidopsis. Example: gene,Xa21. Weigel et al. Cell wall- Seo et al. (2000). Plant associated(1995). Science Physiol 122, protein kinase 270, 1988-1992. 1003-1014.WAK1. Tobacco Activation Example: MAP kinase: a tagging in Thionins.possible Arabidopsis. mediator in Epple and wound signal Bohlmanntransduction (1997). Plant pathways. Cell 9, 509-520. He et al.Overexpression (1998). Plant J. of an 14, 55-63. endogenous Requirementthionin for the induced enhances expression of a resistance of cell wallArabidopsis associated against receptor kinase Fusarium for survivaloxysporum. during the He et al. pathogen (1998). Plant response. J. 14,55-63. He et al. Requirement (1999). Plant for the Mol. Biol. 39,induced 1189-1196. A expression of cluster of five a cell wall cellwall- associated associated receptor receptor kinase kinase for genes,Wak1-5, survival are expressed during the in specific pathogen organs ofresponse. Arabidopsis. Epple and Bohlmann (1997). Plant Cell 9, 509-520.Overexpression of an endogenous thionin enhances resistance ofArabidopsis against Fusarium oxysporum. Ichimura et al. (1998). DNA Res.5,341-5348. Molecular cloning and characterization of three cDNAsencoding putative mitogen- activated protein kinase kinases (MAPKKs) inArabidopsis thaliana. 2. Stress Manipulate Close, T. J. Test for Winkleret al. response to drought (1996). enhanced (1998). Plant cold, drought,resistance. Physiol. Plant sensitivity to Physiol. 118, salinity, seedManipulate 97, 795-803. drought, 743-750. maturation, desiccationDehydrins: dessication, Systematic embryo tolerance in emergence ofcold, salinity, reverse development, flowers, a biochemical in ovules,genetics of ABA. ovules and role of a developing transfer- Example:seeds. family of seed and DNA-tagged Dehydrins Manipulate plantseedlings. lines of Example: cold tolerance dehydration Test forArabidopsis. NPK1-like in flowers, proteins. enhanced Weigel et al.protein kinase ovules, and Kovtun et al. tolerance to (2000). PlantExample: seeds. (2000). PNAS drought, Physiol 122, DNA bindingManipulate USA 97, dessication, 1003-1014. protein genes: seed2940-2945. cold, salinity, Activation CBF-like, dormancy. Functional inovules, tagging in DREB2A, Manipulate analysis of developingArabidopsis. RAP2.1. germination oxidative seed and seed. frequency.stress- Test for Manipulate activated changes in seed storage mitogen-seed viability and viability. activated upon storage. protein kinaseTest for cascade in changes in plants. germination frequencies. 3.Response Altered Bender and Test for Winkler et al. to starvation,response to Fink (1998). enhanced (1998). Plant wounding, starvation. Amyb sensitivity to Physiol. 118, and pathogen Altered homologue,starvation, 743-750. attack by response to ATR1, wounding, Systematictryptophan wounding. activates and pathogen reverse synthesis. Alteredtryptophan attack. genetics of Example: response to gene Test fortransfer- DNA binding pathogen expression in enhanced DNA-taggedprotein; attack. arabidopsis. tolerance to lines of ATR1-like PNAS USAstarvation, Arabidopsis. myb. 95, 5655-5660. wounding, Weigel et al.Example: and pathogen (2000). Plant Auxin attack. Physiol 122,conjugating 1003-1014. enzyme; Activation indole-3- tagging in acetatebeta- Arabidopsis. glucosyltransferase. Cell Stearoyl-acyl Production ofMerlo et al. Analyze seed Winkler et al. metabolism carrier oils high in(1998). Plant size. (1998). Plant protein saturated fatty Cell 10,1603-1621. Analyze seed Physiol. 118, desaturase acids yield. 743-750.Example: Manipulate Analyze seed Systematic C18 fatty acid membrancecomposition. reverse desaturation composition Analyze seed genetics ofoil by gas transfer- chromatography. DNA-tagged lines of Arabidopsis.Weigel et al. (2000). Plant Physiol 122, 1003-1014. Activation taggingin Arabidopsis. Browse et al. (1986). Anal. Biochem. 152, 141-145. 2.Manipulate Manipulate Mathews and Analyze seed Winkler et al. nitrogenasparagine Van Holde size. (1998). Plant economy degradation in Analyzeseed Physiol. 118, Example: ovules and yield. 743-750. Asparaginaseseeds. Analyze seed Systematic Manipulate composition. reverse endospermgenetics of production. transfer- Manipulate DNA-tagged embryo lines ofdevelopment. Arabidopsis. Manipulate Weigel et al. ovule size. (2000).Plant Manipulate Physiol 122, seed size. 1003-1014. Activation taggingin Arabidopsis. Heath et al. (1986). Planta 169, 304-312. Browse et al.(1986). Anal. Biochem. 152, 141-145. D'Aoust et al. (1999). Plant Cell11, 2407-2418.

Fruit development responsive polynucleotides are characteristicallydifferentially transcribed in response to fluctuatingdevelopmental-specific polynucleotide levels or other signals, whetherinternal or external to a cell. MA_diff reports the changes intranscript levels of various fruit development responsivepolynucleotides in fruits.

These data can be used to identify a number of types of fruitdevelopment responsive polynucleotides. Profiles of some of thesedifferent fruit development responsive polynucleotides are shown in thetable below with examples of the kinds of associated biologicalactivities. Because development is a continuous process and many celltypes are being examined together, the expression profiles of genesoverlap between stages of development in the chart below.

Examples of Developmental Biochemical Transcript Levels ProcessMetabolic Pathways Activity (0-5 mm) >> (5-10 mm) ≅ Ovule ElongationHormone Production, Transcription (>10 mm) Tissue SpecializationTransport, Perception, Factors (0-5 mm) >> (5-10 mm) > Vascular systemSignalling, Response (e.g., Transporters (>10 mm) Meristem Gibberellin,Ethylene, Auxin) Kinases (0-5 mm) > (5-10 mm) ≅ Endosperm Cell wallBiosynthesis Changes in (>10 mm) Seed coat Lipid Biosynthesiscytoskeletal Fruit Specific Gene Transcription protein activityInitiation modulating cell Sucrose Mobilization and structurePartitioning Stability factors Sucrose Signaling for proteinLipoxygenase translation Localization Changes in cell Repressors ofMetabolic wall/membrane Pathways structure Protein Remodeling Chromatinstructure and/or DNA topology Biosynthetic enzymes (5-10 mm) >> (0-5mm) > Tissue Specialization Cell Wall Biosynthesis Transcription (>10mm) Vascular System Specific Gene Transcription Factors (5-10 mm) > (0-5mm) ≅ Organelle Initiation Transporters (>10 mm) Differentiation SucroseMobilization and Kinases (5-10 mm) >> (0-5 mm) ≅ Cotyledon ElongationPartitioning Chaperones (>10 mm) (cell division) Sucrose SignalingChanges in Vacuome Repressors of Metabolic cytoskeletal DevelopmentPathways protein activity Lipid Deposition Auxin Perception, modulatingcell Response and Signaling strucure Protein Remodeling Stability ofLipid Biosynthesis and factors for Storage protein translation Changesin cell wall/membrane structure Chromatin structure and/or DNA topologyBiosynthetic enzymes (>10 mm) > (0-5 mm) ≅ Cotyledon Elongation CellElongation Transcription (5-10 mm) (expansion) Specific GeneTranscription Factors Lipid Deposition Initiation Transporters ProteinDeposition Sucrose Mobilization and Kinases Desiccation PartitioningChaperones Sucrose Signaling for protein Lipoxygenase translationLocalization Changes in cell Repressors of metabolic wall/membranepathways structure Hormone Perception, Chromatin Response and Signaling(e.g. structure and/or abscissic acid) DNA topology Protein RemodelingBiosynthetic Protein synthesis and Storage enzymes Lipid Synthesis andStorage Metabolic Acquisition of Dessication enzymes ToleranceSenescence (0-5 mm) < (5-10 mm) ≅ Ovule Elongation Cell elongationTranscription (>10 mm) Repressors of Negative regulation of Factors (0-5mm) << (5-10 mm) ≅ Ethylene ethylene pathways Transporters (>10 mm)production Maintenance of Ethylene Kinases (0-5 mm) << (5-10 mm) <Tissue response Chaperones (>10 mm) specialization Changes in pathwaysand Stability of (0-5 mm) << (>10 mm) < Vascular System processesoperation in cells factors (5-10 mm) Meristem Biosynthetic Cotyledonenzymes Seed Coat Metabolic enzymes (5-10 mm) < (0-5 mm) ≅ OrganelleNegative regulation of Transcription (>10 mm) differentiation hormonepathways Factors Cotyledon elongation Maintenance of hormoneTransporters (division) response Kinases Vacuome Changes in pathways andChaperones development processes operation in cells Lipid developmentDehydration and acquisition of Desiccation desiccation toleranceSenescence (>10 mm) < (0-5 mm) ≅ Cotyledon Elongation Cell elongationTranscription (5-10 mm) (expansion) Negative regulation of Factors Lipiddeposition hormone pathways Transporters Protein deposition Maintenanceof hormone Kinases Desiccation response Chaperones Changes in pathwaysand Metabolic processes operation in cells enzymes Dehydration andacquisition of Biosynthetic desiccation tolerance enzymes Senescence(0-5 mm) ≅ (5-10 mm) ≅ All stages Ribosome/polysome Transcription (>10mm) production and maintenance Factors Housekeeping genes TransportersKinases Chaperones

III.B. Development Genes, Gene Components and Products

III.B.1. Imbibition and Germination Responsive Genes, Gene Componentsand Products

Seeds are a vital component of the world's diet. Cereal grains alone,which comprise ˜90% of all cultivated seeds, contribute up to half ofthe global per capita energy intake. The primary organ system for seedproduction in flowering plants is the ovule. At maturity, the ovuleconsists of a haploid female gametophyte or embryo sac surrounded byseveral layers of maternal tissue including the nucleus and theinteguments. The embryo sac typically contains seven cells including theegg cell, two synergids, a large central cell containing two polarnuclei, and three antipodal cells. That pollination results in thefertilization of both egg and central cell. The fertilized egg developsinto the embryo. The fertilized central cell develops into theendosperm. And the integuments mature into the seed coat. As the ovuledevelops into the seed, the ovary matures into the fruit or silique.Late in development, the developing seed ends a period of extensivebiosynthetic and cellular activity and begins to desiccate to completeits development and enter a dormant, metabolically quiescent state. Seeddormancy is generally an undesirable characteristic in agriculturalcrops, where rapid germination and growth are required. However, somedegree of dormancy is advantageous, at least during seed development.This is particularly true for cereal crops because it preventsgermination of grains while still on the ear of the parent plant(preharvest sprouting), a phenomenon that results in major losses to theagricultural industry. Extensive domestication and breeding of cropspecies have ostensibly reduced the level of dormancy mechanisms presentin the seeds of their wild ancestors, although under some adverseenvironmental conditions, dormancy may reappear. By contrast, weed seedsfrequently mature with inherent dormancy mechanisms that allow someseeds to persist in the soil for many years before completinggermination.

Germination commences with imbibition, the uptake of water by the dryseed, and the activation of the quiescent embryo and endosperm. Theresult is a burst of intense metabolic activity. At the cellular level,the genome is transformed from an inactive state to one of intensetranscriptional activity. Stored lipids, carbohydrates and proteins arecatabolized fueling seedling growth and development. DNA and organellesare repaired, replicated and begin functioning. Cell expansion and celldivision are triggered. The shoot and root apical meristem are activatedand begin growth and organogenesis. Schematic 4 summarizes some of themetabolic and cellular processes that occur during imbibition.Germination is complete when a part of the embryo, the radicle, extendsto penetrate the structures that surround it. In Arabidopsis, seedgermination takes place within twenty-four (24) hours after imbibition.As such, germination requires the rapid and orchestrated transcriptionof numerous polynucleotides. Germination is followed by expansion of thehypocotyl and opening of the cotyledons. Meristem development continuesto promote root growth and shoot growth, which is followed by early leafformation.

Genes with activities relevant to imbibition-germination and earlyseedling growth are described in the two sections A and B below.

III.B.1.a. Identification of Imbibition and Germination Genes

Imbibition and germination includes those events that commence with theuptake of water by the quiescent dry seed and terminate with theexpansion and elongation of the shoots and roots. The germination periodexists from imbibition to when part of the embryo, usually the radicle,extends to penetrate the seed coat that surrounds it. Imbibition andgermination genes identified herein are defined as genes, genecomponents and products capable of modulating one or more processes ofimbibition and germination described above. They are useful to modulatemany plant traits from early vigor to yield to stress tolerance.Examples of such germination genes and gene products are shown in theReference and Sequence Tables. The functions of many of the genes werededuced from comparisons with known proteins and are also given in theREF Tables.

Imbibition and Germination Genes Identified by Phenotypic Observations

Imbibition and germination genes are active, potentially active or moreactive during growth and development of a dry seed into a seedling.These genes herein were discovered and characterized from a much largerset of genes in experiments designed to find genes that cause poorgermination.

In these experiments, imbibition and germination genes were identifiedby either 1) ectopic expression of a cDNA in a plant or (2) mutagenesisof the plant genome. The seeds were then imbibed and cultivated understandardized conditions and any phenotypic differences in the modifiedplants compared with the parental “wild-type” seedlings were recorded.The genes causing the changes were deduced from the cDNA inserted orgene mutated. The phenotypic differences observed were poor germinationand aberrant seedlings.

Imbibition and Germination Genes Identified by Differential Expression

Germination genes were also identified by measuring the relative levelsof mRNA products of genes in different stages of germination of a seedversus the plant as a whole. Specifically, mRNA was isolated from wholeimbibed seeds of Arabidopsis plants 1, 2, 3 or 4 days after imbibitionand compared to mRNA isolated from dry seed-utilizing microarrayprocedures. The MA_diff Table reports the transcript levels of theexperiment. For transcript levels that were higher in the imbibed seedthan in dry seed a “+” is shown. A “−” is shown when the transcriptlevels in dry seed were greater than those in imbibed seed. For moreexperimental detail, see the examples below:

Germination associated genes can be identified by comparing expressionprofiles of imbibed gibberellin treated and untreated gal mutant seed.Germination associated genes can also be identified by comparingexpression profiles in late maturation seed from wild-type and mutantsthat are defective for the establishment of dormancy and can germinateprecociously (e.g. aba1, aba2, abi4 in arabidopsis and vp1, vp5 inmaize) or are defective for the specification of cotyledon identity anddessication tolerance (e.g. lec1, lec2, and fus3).

The MA_diff Table(s) reports the transcript levels of the experiment(see EXPT ID: 108461, 108462, 108463, 108464, 108528, 108529, 108530,108531, 108545, 108546, 108547, 108518, 108529, 108543, 108544). Fortranscripts that had higher levels in the samples than the control, a“+” is shown. A “−” is shown for when transcript levels were reduced inroot tips as compared to the control. For more experimental detail seethe Example section below.

Imbibed & Germinating Seeds genes are those sequences that showeddifferential expression as compared to controls, namely those sequencesidentified in the MA_diff tables with a “+” or “−” indication.

Imbibed & Germinating Seeds Genes Identified by Cluster Analyses ofDifferential Expression

Imbibed & Germinating Seeds Genes Identified by Correlation to Genesthat are Differentially Expressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of Imbibed & Germinating Seeds genes is any groupin the MA_clust that comprises a cDNA ID that also appears in Expt ID108461, 108462, 108463, 108464, 108528, 108529, 108530, 108531, 108545,108546, 108547, 108518, 108529, 108543, 108544 of the MA_diff table(s).

Imbibed & Germinating Seeds Genes Identified by Correlation to Genesthat Cause Physiological Consequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of Imbibed &Germinating Seeds genes. A group in the MA_clust is considered a Imbibed& Germinating Seeds pathway or network if the group comprises a cDNA IDthat also appears in Knock-in or Knock-out tables that causes one ormore of the phenotypes described in section above.

Imbibed & Germinating Seeds Genes Identified by Amino Acid SequenceSimilarity

Imbibed & Germinating Seeds genes from other plant species typicallyencode polypeptides that share amino acid similarity to the sequencesencoded by corn and Arabidopsis Imbibed & Germinating Seeds genes.Groups of Imbibed & Germinating Seeds genes are identified in theProtein Group table. In this table, any protein group that comprises apeptide ID that corresponds to a cDNA ID member of a Imbibed &Germinating Seeds pathway or network is a group of proteins that alsoexhibits Imbibed & Germinating Seeds functions/utilities.

III.B.1.b. Use of Imbibition and Germination Genes, Gene Components andProducts to Modulate Phenotypes

Imbibition and germination genes and gene products can be divided intothose that act during primary events, secondary events, and/ortermination. The genes and gene products of the instant invention areuseful to modulate any one or more of the phenotypes described below:

I. Primary Events

-   -   A. Dormancy

Imbibition and germination genes and gene products of the invention canact to modulate different types of dormancy including:

-   -   1. Primary dormancy—dormancy is established during seed        development    -   2. Seed coat-imposed dormancy—dormancy is imposed by blocking        water uptake, mechanical restraint of embryo, blocking the exit        of inhibitors    -   3. Embryo dormancy—cotyledon mediated inhibition of embryonic        axis growth    -   4. Secondary dormancy—dormancy is induced when dispersed, mature        seeds are exposed to unfavorable conditions for germination        (e.g. anoxia, unsuitable temperature or illumination).    -   5. Hormone-induced

B. Dormancy-Breaking Signal Perception and Transduction

Germination genes and gene products include those that are able tomodulate the response to dormancy releasing signals such as fruitripening and seed development; imbibition; temperature (low and high,range 0-23°); light, particularly for coat imposed dormancy (whitelight, intermittent illumination, orange and red region of the spectrum(longer than 700 or 730 nm), and phytochrome); coat softening; chemicals(respiratory inhibitors, sulfhydryl compounds, oxidants, nitrogenouscompounds, growth regulators—ga, ba, ethylene, and various, ethanol,methylene blue, ethyl ether, fusicoccin); oxygen and carbon dioxide; andstress.

II. Secondary Events

During the secondary events of germination, dormancy-maintainingmetabolism is repressed, dormancy-breaking metabolism is induced andstructures surrounding the embryo weaken (where present). Germinationgenes and gene products are useful to modulate processes of thesecondary events including water uptake, such as cell expansion andchange in osmotic state (ion exchange); and respiration—(oxygenconsumption). the genes and genes products of the invention can regulatethe following pathways which resume during the first respiratory burstof germination including glycolysis, pentose phosphate, citric acid, andtricarboxylic acid cycle.

A. Mitochondrial Development

Tissues of the mature dry seed contain mitochondria, and although theseorganelles are poorly differentiated as a consequence of the drying,they contain sufficient Kreb's cycle enzymes and terminal oxidases toprovide adequate amount of ATP to support metabolism for several hoursafter imbibition. During germination of embryos, there appears to be twodistinct patterns of mitochondrial development. In starch-storing seeds,repair and activation of preexisting organelles predominate, whereasoil-storing seeds typically produce new mitochondria. Germination genesand gene products of the invention are useful to modulate the repair,activation and biogenesis pathways of mitochondria, including membraneformation and repair, DNA repair and synthesis, protein synthesis, andcoordinated regulation of mitochondrial and nuclear genomes

B. Metabolism

In addition to respiration and organelle activity, enzyme activity, DNArepair, RNA synthesis and protein synthesis are fundamental cellularactivities intimately involved in the completion of germination and thepreparation for subsequent growth. Imbibition and germination genes andgene products of the invention can participate in or modulate theseactivities, including ABA response, GA response, ATP synthesis andadenylate energy charge during germination, and the synthesis andutilization of reducing power: pyridine nucleotides (NADH and NADPH)

III. Termination

The last stage of seed germination is characterized by the emergence ofthe radicle or root apex through the seed coat. Typically, the cellwalls loosen and the radicle extends from the embryo during lategermination. Germination genes and gene products are useful to modulatethe mobilization of stored reserves, DNA synthesis and cell divisionthat are typical of this stage of germination.

To regulate any of the phenotype(s) above, activities of one or more ofthe late germination genes or gene products can be modulated and testedby screening for the desired trait. Specifically, the gene, mRNA levels,or protein levels can be altered in a plant utilizing the proceduresdescribed herein and the phenotypes can be assayed. As an example, aplant can be transformed according to Bechtold and Pelletier (1998,Methods. Mol. Biol. 82:259-266) and/or screened for variants as inWinkler et al. (1998) Plant Physiol 118: 743-50 and visually inspectedfor the desired phenotype or metabolically and/or functionally assayedaccording to Dolan et al. (1993, Development 119: 71-84), Dolan et al.(1997, Development 124: 1789-98), Crawford and Glass (1998, Trends PlantScience 3: 389-95), Wang et al. (1998, PNAS USA 95: 15134-39), Gaxiolaet al. (1998, PNAS USA 95: 4046-50), Apse et al. (1999, Science 285:1256-58), Fisher and Long (1992, Nature 357: 655-60), Schneider et al.(1998, Genes Devel 12: 2013-21) and Hirsch (1999, Curr Opin Plant Biol.2: 320-326).

III.B.1.c. Use of Imbibition and Germination Genes, Gene Components andProduct to Modulate Biochemical Activities

The roles of the biochemical changes associated with imbibition andgermination can be appreciated from a summary of the processesoccurring.

Physiology

Water plays an important role throughout the plant life cycle. The mostdramatic example of this is in seed germination. Although germination istriggered by water, the germination response is also positivelyregulated by the plant growth regulators the gibberellins and negativelyaffected by the growth regulator abscisic acid. Genes that are activatedby water and genes that are activated by gibberellins can be identifiedthrough expression profiling experiments using arabidopsis mutantsdefective for gibberellin biosynthesis or perception (gal, gai),abscisic acid biosynthesis or perception (aba1, abi3, and abi4) in thepresence or absence of exogenous gibberellins. These genes can be usedto promote seedling growth and development and other phases of plantdevelopment.

Transcriptional Control of Gene Activity

At the end of seed development, dessication and dormancy have imposed aglobal state of repression on gene activity throughout the seed.Reactivation of the genome requires water and gibberellins. One functionof the genes that are activated early by imbibition is the rapid anddramatic reversal of gene repression. For example, expression-profilingexperiments revealed that several thousand genes are hyperactivated inarabidopsis upon imbibition. These include genes involved in metabolicpathways, genes that promote cell growth and division, andtranscriptional control genes. Thus one class of genes expressed earlyin imbibition includes those that promote high levels of geneexpression. Other early genes are responsible for regulating specificmetabolic, cell, and developmental processes. The strategy fordistinguishing these functions was outlined in the Introduction.

Mobilization of Storage Reserves

In contrast to the synthesis and accumulation of reserves during seeddevelopment an important function of genes expressed during imbibitionand germination is the control of the mobilization and catabolism ofseed storage reserves in the endosperm (in grasses and cereals) and theembryo. The mobilization of seed storage reserves is triggered byimbibition and may occur over several days. There are three classes ofhigh molecular weight seed storage reserves: carbohydrates,triacylglycerols, and storage proteins. Upon imbibition seed storagereserves are converted into forms that can be transported andmetabolized. Genes encoding enzymes for storage reserve catabolism areexpressed shortly after imbibition. Starch for example is converted tosucrose. Triacylglycerols are converted into acetyl-CoA. Storageproteins are converted into amino acids or deaminated to provide carbonskeletons for oxidation.

Carbohydrate Catabolism

Starch is the most common storage carbohydrate in seeds. The primarycomponents of starch are amylose and amylopectin.

Mobilization

There are two pathways for starch catabolism—hydrolytic andphosphorolytic. The product of these pathways is the monosaccharideglucose. Examples of the enzymes responsible for hydrolytic catabolismof starch are: amylase, glucosidase, amylase, dextrinase, isoamylase.The enzyme responsible for phosphorolytic activity is starchphosphorylase.

Transport

The mobilization of starch involves the synthesis of sucrose fromglucose, which can then be transported to sites for growth in the rootand shoot. In some seeds, maltose may be a major form of transportedcarbohydrate. The production of sucrose-6-P from glucose involves thefollowing enzymes: UDP-glucose pyrophosphorylase, sucrose-6-Psynthetase, and sucrose phosphatase.

Sucrose Catabolism

In target tissues sucrose is hydrolyzed by fructofuransidase (invertase)and/or sucrose synthetase. The synthesis of glucose from glucose-1-Pinvolves sucrose synthetase.

Cell Biology

The lumen of the endoplasmic reticulum (ER) is target for otherhydrolase activities including mannosidase, glucosaminidase, acidphosphatase, phosphodiesterase, and phospholipase D.

Triacylglycerol (TAG) Catabolism

Triacylglycerols are the major storage lipids of seeds. The products ofTAG catabolism in imbibed and germinating seed are glycerol and freefatty acids. Most of the glycerol is converted to sucrose for export.Free fatty acids are catabolized through oxidation through theglyoxylate cycle and gluconeogenesis.

Mobilization

Hydrolysis of triacylglycerols is by lipases yielding glycerol and freefatty acids. Free fatty acids are oxidized to acetyl-CoA andpropionyl-CoA via oxidation requiring ATP and coenzyme A. Catabolism ofunsaturated fatty acids also requires cis, trans-isomerases, epimerases,and hydratases. Acetyl-CoA is oxidized through the citric acid cycle toCO2 and H2O. More importantly, acetyl-CoA can be utilized via theglyoxylate cycle and gluconeogenesis for glucose synthesis. Free fattyacids are also broken down via oxidation. Glycerol is converted viaphosphorylation and oxidation to DHAP and G3P, which are used tosynthesize glucose or oxidized via the citric acid cycle. Examples ofother induced enzymes include isocitrate lyase and malate synthetase

Transport

Most of the glycerol, acetyl-CoA, and propionyl-CoA are converted tosucrose for transport. This requires the enzymes glycerol kinase andglycerol phosphate oxidoreductase.

Cell Biology

Glyoxysome biogenesis is required to support fatty acid catabolism andgluconeogenesis. Upon exposure to light there is a loss of glyoxysomesdue to their conversion to peroxisomes.

Storage Protein Catabolism

Mobilization

The hydrolysis of storage proteins to amino acids is performed by adiverse group of proteinases and peptidases. The peptidases includeendopeptidases, aminopeptidases, and carboxypeptidases. They include theA and B class proteinases. The liberated amino acids are available forprotein synthesis, for deamination and reutilization of ammonia viaglutamine and asparagine synthesis, and to provide carbon skeletons forrespiration. Several enzymes including, deaminase, asparaginesynthetase, glutamine synthetase and glutamate dehydrogenase areimportant players in the mobilization and utilization of stored nitrogenin imbibed seed.

Transport

The major transported form of amino acid in germinated seeds isasparagine. In some species glutamine and/or homoserine are the majorform of transported amino acid. Aspartate, glutamate, alanine, glycine,and serine can be converted to sucrose and transported as sucrose. Otheramino acids are transported unchanged.

Cell Biology

Proteinases are sequestered in lumen of endoplasmic reticulum (ER) whichthen fuses with protein bodies.

While catabolism is high in the storage tissues of imbibed seed theproducts of catabolism are transported to sites of growth including theshoot and root apices fueling respiration, biosynthesis, cell divisionand differentiation.

Development

Imbibition triggers several key processes for seedling development. Oneis the activation of the shoot and root apical meristems. The shootapical meristem is responsible for two primary growth activities. One isthe production of the protoderm, procambium and ground meristem. Theprotoderm gives rise to the epidermal system of the plant, theprocambium to the primary vascular tissues, and the ground meristem tothe ground tissues including the cortex and pith. The second is theproduction of leaf primordia, which arise on the flanks of the apex.Thus, activation of the shoot apical meristem results in shoot growthand organogenesis.

The root apical meristem, by contrast is responsible for vegetative rootdevelopment. The first primary growth activity of the root apicalmeristem is the production of the protoderm, procambium and groundmeristem. The second primary growth activity is the production of thecells that give rise to the root cap.

Genes that govern shoot apical meristem activation and development canbe identified in arabidopsis by gene profiling experiments comparinggene expression in wild-type imbibed seed and partial loss-of-functionstm (shootmeristemless) mutants (see SAM). Genes governing root meristemactivity can be identified by gene profiling experiments comparing geneexpression in wild-type imbibed seed and rml (rootmeristemless) mutants.

Genes identified in this way are useful to promote or retard meristemgrowth, modify and strengthen shoot and root development, promote leafdevelopment as described below.

Changes in the concentration of imbibition-germination activatedpolynucleotides result in the modulation of many other polynucleotidesand polynucleotide products. Examples of such activated responsivepolynucleotides and polynucleotide products relative to leaves andfloral stem and to fruits at different development stages are shown inthe Reference and Sequence Tables. These polynucleotides and/or productsare responsible fore effects on traits such as seedling growth, seedlingviability, and seedling vigor. The polynucleotides were discovered byisolating seeds from Arabidopsis wild-type ecotype “Wassilewskija”imbibed for 24 hours, and measuring the mRNAs expressed in them relativeto those in a leaf and floral stem sample and to those in fruits atdifferent developmental stages.

While imbibition-germination activated polynucleotides andpolynucleotide products can act alone, combinations of thesepolynucleotides also affect germination. Useful combinations includedifferent polynucleotides and/or polynucleotide products that havesimilar transcription profiles or similar biological activities, andmembers of the same or functionally similar biochemical pathways. Inaddition, the combination of imbibition germination activatedpolynucleotides and/or polynucleotide products with environmentallyresponsive polynucleotides is also useful because of the interactionsthat exist between development, hormone-regulated pathways, stress andpathogen induced pathways and nutritional pathways. Here, usefulcombinations include polynucleotides that may have differenttranscription profiles, and participate in common or overlappingpathways but combine to produce a specific, phenotypic change.

Such imbibition and germination activated polynucleotides andpolynucleotide products can function to either increase or dampen theabove phenotypes or activities either in response to transcript changesin fruit development or in the absence of fruit-specific polynucleotidefluctuations.

BIOCHEMICAL OR METABOLIC ACTIVITIES AND/OR PATHWAYS CITATIONS INCLUDINGPROCESS ALTERED ASSAYS Growth, Differentiation Farnesylation MediatedSeed Pei et al (1998) Science 282: and Development Dormancy 287-290;Cutler et al. (1996) Science 273: 1239 Metabolic activity Nitrogenmetabolism Goupil et al (1998) J Exptl Botany 49: 1855-62 Metabolicactivity —H+ export and membrane Cerana et al. (1983) hyperpolarizationMetabolic activity Chloroplast functioning Benkova et al (1999) PlantPhysil 121: 245-252 Growth, Differentiation Regulation of MorphogenesisRiou-Khamlichi et al. (1999) and development Science 283: 1541-44Metabolic activity Cell Death Lohman et al. (1994) Physiol Plant 92:322-328 Growth and development Promotion of cell division Kakimoto(1996) Science Shoot formation in absence of 274: 982-985 exogenouscytokinin Metabolic activity Membrane repair Heath et al. (1986) Planta169: 304-12 Browse et al. (1986) Anal Biochem 152: 141-5 D'Aoust et al(1999) Plant Cell 11: 2407-18 Metabolism Organic molecule export Moodyet al. (1988) Phytochemistry 27: 2857-61 Metabolic activity NutrientUptake Uozumio et al. (2000) Plant Physiol 122: 1249-59 Metabolicactivity Ion export Uozumi et al. (2000) Plant Physiol 122: 1249-59Frachisse et al. (2000) Plant J 21: 361-71 Growth, DifferentiationDivision and/or elongation Zhang and Forde (1998) and developmentScience 279: 407-409. Coruzzi et al. U.S. Pat. No. 5,955,651 Metabolicactivity Regulation of Molecular Wisniewski et al. (1999) chaperonesPhysiolgia Plantarum 105: 600-608 Metabolic activity Reactivation ofAggregation Lee and Vierling (2000) Plant and Protein Folding Physiol.122: 189-197 Metabolic activity Maintenance of Native Queitsch et al.(2000) The Conformation (cytosolic proteins) Plant Cell 12: 479-92Metabolic activity Regulation of Translational Wells et al. (1998) Genesand Efficiency Development 12: 3236-51 Metabolic activity DNA RepairBewley (1997) Plant Cell 9: 1055-66 Metabolic activity Protein Synthesisusing stored Heath et al. (1986) Planta 169: or newly synthesized mRNAs304-12 Metabolic activity Mitochondrial repair and MacKenzie andMcIntosh synthesis (1999) Plant Cell 11: 571-86 Metabolic activityCommencement of respiration Debeaujon et al. (2000) Plant Physiol 122:403-4132 Water Uptake Debeaujon et al. (2000) Plant Physiol 122:403-4132

Other biological activities that are modulated by theimbibition-activated polynucleotides and polynucleotide products arelisted in the Reference Tables. Assays for detecting such biologicalactivities are described in the Table below as well as in the Domainsection of the Reference Table.

III.B.1.d. Use of Imbibition and Germination Genes to Modulate theTranscription Levels of Other Genes

The expression of many genes is “upregulated” or “downregulated” duringimbibition and germination because some imbibition and germination genesare integrated into complex networks that regulate transcription of manyother genes. Some imbibition and germination genes are therefore usefulfor regulating other genes and hence complex phenotypes.

Imbibition-activated polynucleotides may also be differentiallytranscribed in response to fluctuating developmental-specificpolynucleotide levels or concentrations, whether internal or external toa cell, at different times during the plant life cycle to promoteassociated biological activities. These activities are, by necessity, asmall subset of the genes involved in the development process.Furthermore, because development is a continuous process with few cleardemarcations between stages, the associated metabolic and biochemicalpathways overlap. Some of the changes in gene transcription aresummarized in the Table below:

EXAMPLES OF BIOCHEMICAL REGULATORY DEVELOPMENTAL PHYSIOLOGICAL/METABOLICACTIVITIES PROCESS REGULATED CONSEQUENCES OF ASSOCIATED WITH BYIMBIBITION- MODIFYING GENE IMBIBITION AND GERMINATION GENES PRODUCTLEVELS GERMINATION Tissue Specialization Lipid Catabolism TranscriptionFactors Cotyledon Expansion Lipoxygenase Transporters Endosperm (???)Localization Kinases Activation of the Shoot Starch Catabolism Changesin cytoskeletal Apical Meristem Seed Protein Catabolism protein activityActivation of the Root Growth Regulator Production, modulating cellstructure Apical Meristem Transport, Perception, Stability of factorsfor Radicle Growth Signaling, Response (e.g., protein translationVascular System Gibberellins, Ethylene, Changes in cell DevelopmentAuxin) wall/membrane structure Global Gene Activation Chromatinstructure Transcription Initiation and/or DNA topology Sucrose Synthesisand Biosynthetic enzymes Partitioning Metabolic enzymes Sucrosecatabolism Sucrose Signaling Cell Wall Biosynthesis Activators ofMetabolic Pathways Protein Remodeling Organelle Differentiation CellWall Biosynthesis Transcription Factors and Development Membrane Repairand Transporters Synthesis Kinases Specific Gene TranscriptionChaperones Initiation Changes in cytoskeletal Sucrose Mobilization andprotein activity Partitioning modulating cell structure SucroseSignaling Stability of factors for Activators of Metabolic proteintranslation Pathways Changes in cell Auxin Perception, wall/membranestructure Response and Signaling Chromatin structure Protein Remodelingand/or DNA topology Lipid Mobilization, Biosynthetic enzymes Metabolismand Biosynthesis Metabolic enzymes Protein Transport, Metabolism, andBiosynthesis DNA Repair Cell Division Transcription Factors Cell CycleControl Transporters DNA Replication Kinases Specific Gene TranscriptionChaperones Initiation for protein translation Protein Remodeling Changesin cell Protein Synthesis wall/membrane structure Repressors ofSenescence Chromatin structure and/or DNA topology Biosynthetic enzymesCellular Metabolism Lipid Catabolism Transcription Factors oxidationTransporters Glyoxylate cycle Kinases Citric acid cycle ChaperonesGluconeogenesis Translation Initiation Sucrose Synthesis and FactorsPartitioning Biosynthetic Enzymes Starch Catabolism Metabolic EnzymesSeed Protein Catabolism Asparagine Synthesis and Transport Sucrosecatabolism Sucrose Signaling Ribosome/polysome production andmaintenance Housekeeping genes Respiration Photosynthesis

Changes in the processes of germination are the result of modulation ofthe activities of one or more of these many germination genes and geneproducts. These genes and/or products are responsible for effects ontraits such as fast germination, plant vigor and seed yield, especiallywhen plants are growing in the presence of biotic or abiotic stresses orwhen they are growing in barren conditions or soils depleted of certainminerals.

Germination genes and gene products can act alone or in combination asdescribed in the introduction. Of particular interest are combination ofthese genes and gene products with those that modulate stress toleranceand/or metabolism. Stress tolerance and metabolism genes and geneproducts are described in more detail in the sections below.

Use of Promoters of Imbibition and Germination Genes

These promoters can be used to control expression of any polynucleotide,plant or non-plant, in a plant host. Selected promoters when operablylinked to a coding sequence can direct synthesis of the protein inspecific cell types or to loss of a protein product, for example whenthe coding sequence is in the antisense configuration. They are thususeful in controlling changes in imbibition and germination phenotypesor enabling novel proteins to be made in germinating seeds.

III.B.2. Early Seedling-Phase Specific Responsive Genes, Gene Componentsand Products

One of the more active stages of the plant life cycle is a few daysafter germination is complete, also referred to as the early seedlingphase. During this period the plant begins development and growth of thefirst leaves, roots, and other organs not found in the embryo. Generallythis stage begins when germination ends. The first sign that germinationhas been completed is usually that there is an increase in length andfresh weight of the radicle.

III.B.2.a. Identification of Early Seedling Phase Genes, Gene Componentsand Products

These genes defined and identified herein are capable of modulating oneor more processes of development and growth of many plant organs asdescribed below. These genes and gene products can regulate a number ofplant traits to modulate yield. Examples of such early seedling phasegenes and gene products are shown in the Reference and Sequence,Knock-in, Knock-out and MA-diff Tables. The functions of the protein ofsome of these genes are also given in these Tables.

Early Seedling Genes Identified by Phenotypic Observations

Some early seedling genes were discovered and characterized from a muchlarger set of genes by experiments designed to find genes that causephenotypic changes in germinating seeds as the transitioned intoseedlings.

In these experiments, leaf genes were identified by either (1) ectopicexpression of a cDNA in a plant or (2) mutagenesis of the plant genome.The plants were then cultivated and one or more of the following leafphenotypes, which varied from the parental “wild-type”, were observed:

-   -   Abnormal growth    -   Abnormal cotyledons or root growth        -   Reduced growth        -   Abnormal first leaf        -   Abnormal hypocotyl        -   Abnormal pigmentation            The genes identified by these phenotypes are given in the            Knock-in and Knock-out Tables.

Early Seedling Phase Genes Identified by Differential Expression

Such genes are active or potentially active to a greater extent indeveloping and rapidly growing cells, tissues and organs, as exemplifiedby development and growth of a seedling 3 or 4 days after planting aseed. These genes herein were also discovered and characterized from amuch larger set of genes in experiments designed to find genes. Earlyseedling phase genes were identified by measuring the relative levels ofmRNA products in a seedling 3 or 4 days after planting a seed versus asterilized seed. Specifically, mRNA was isolated from aerial portion ofa seedling 3 or 4 days after planting a seed and compared to mRNAisolated from a sterilized seed utilizing microarray procedures. TheMA_diff Table(s) reports the transcript levels of the experiment (seeEXPT ID: Sqn (relating to SMD 7133, SMD 7137)). For transcripts that hadhigher levels in the samples than the control, a “+” is shown. A “−” isshown for when transcript levels were reduced in root tips as comparedto the control. For more experimental detail see the Example sectionbelow.

Early Seedling Phase genes are those sequences that showed differentialexpression as compared to controls, namely those sequences identified inthe MA_diff tables with a “+” or “−” indication.

Early Seedling Phase Genes Identified by Cluster Analyses ofDifferential Expression

Early Seedling Phase Genes Identified by Correlation to Genes that areDifferentially Expressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of Early Seedling Phase genes is any group in theMA_clust that comprises a cDNA ID that also appears in Expt ID Sqn(relating to SMD 7133, SMD 7137) of the MA_diff table(s).

Early Seedling Phase Genes Identified by Correlation to Genes that CausePhysiological Consequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of EarlySeedling Phase genes. A group in the MA_clust is considered a EarlySeedling Phase pathway or network if the group comprises a cDNA ID thatalso appears in Knock-in or Knock-out tables that causes one or more ofthe phenotypes described in section above.

Early Seedling Phase Genes Identified by Amino Acid Sequence Similarity

Early Seedling Phase genes from other plant species typically encodepolypeptides that share amino acid similarity to the sequences encodedby corn and Arabidopsis Early Seedling Phase genes. Groups of EarlySeedling Phase genes are identified in the Protein Group table. In thistable, any protein group that comprises a peptide ID that corresponds toa cDNA ID member of a Early Seedling Phase pathway or network is a groupof proteins that also exhibits Early Seedling Phase functions/utilities.

Of particular interest are early seedling phase genes that aredifferentially expressed 3 or 4 days after planting a seed but notdifferentially expressed germinating seeds and/or mature leaves.

Examples of phenotypes, biochemical activities, and transcriptionprofiles that can be modulated by these genes and gene products aredescribed above and below.

III.B.2.b. Use of Early Seedling Genes, Gene Components and Products toModulate Phenotypes

Rapid, efficient establishment of a seedling is very important incommercial agriculture and horticulture. It is also vital that resourcesare approximately partitioned between shoot and root to facilitateadaptive growth. Phototropism and geotropism need to be established. Allthese require post-germination process to be sustained to ensure thatvigorous seedlings are produced. Early seedling phase genes, genecomponents and products are useful to manipulate these and otherprocesses.

I. Development

The early seedling phase genes, gene components and products of theinstant invention are useful to modulate one or more processes of thestages of leaf morphogenesis including: stage 1—organogenesis that givesrise to the leaf primordium; stage 2—delimiting basic morphologicaldomains; and stage 3—a coordinated processes of cell division,expansion, and differentiation. Early seedling phase genes include thosegenes that terminate as well as initiate leaf development. Modulatingany or all of the processes leads to beneficial effects at specificlocations.

Gene Sequences Affecting Types of Leaves—Applicants provide with thesegenes, gene components and gene products the means to modulate one ormore of the types of leaves, and stem, including cotyledons and majorleaves.

Gene sequences affecting cell properties—These genes, gene componentsand gene products are useful to modulate changes in cell size, celldivision, rate and direction, cell elongation, cell differentiation,xylem and phloem cell numbers, cell wall composition, and all celltypes.

Gene Sequences Affecting Leaf Architecture—Modifying leaf architectureis useful to modulate change in overall leaf architecture includingveination, such as improvements in photosynthetic efficiency, stresstolerance efficiency of solute and nutrient movement to and from theleaf which are accomplished by increases or decreases in vein placementand number of cells in the vein and shape, such as elongated versusrounded and symmetry (around either abaxial-adaxial (dorsiventral) axisor apical-basal (proximodistal) axis, margin-blade-midrib (lateral)axis).

Genes Sequences Influencing Leaf Responses—Shoot apical meristem cellsdifferentiate to become leaf primordia that eventually develop intoleaves. The genes, gene components and gene products of this inventionare useful to modulate any one or all of these growth and developmentprocesses, by affecting timing and rate or planes of cell divisions forexample, in response to the internal plant stimuli and/or programs suchas embryogenesis, germination, hormones (like Auxin), phototropism,coordination of leaf growth and development with that of other organs(like roots and stems), and stress-related program.

II. Interaction with the Environment

Successful seedling establishment demands successful interaction withthe environment in the soil. Early vegetation genes orchestrate andrespond to interactions with the environment. Thus early seedling phasegenes are useful for improving interactions between a plant and theenvironment including pigment accumulation, oxygen gain/loss control,carbon dioxide gain/loss control, water gain/loss control, nutrienttransport, light harvesting, chloroplast biogenesis, circadian rhythmcontrol, light/dark adaptation, defense systems against biotic andabiotic stresses, metabolite accumulation, and secondary metaboliteproduction

III. Organizing Tissues for Photosynthesis and Metabolism

Following germination and utilization of seed reserves, plant tissuesprepare for photosynthesis and seedling metabolism. Leaf meristems, androot meristems participate in these changes before cell differentiation.Many of the uses for plants depend on the success of leaves as thepowerhouses for plant growth, their ability to withstand stresses andtheir chemical composition. Leaves are organs with many different celltypes and structures. Most genes of a plant are active in leaves andtherefore leaves have very diverse of pathways and physiologicalprocesses. Examples of such pathways and processes that are modulated byearly seedling phase genes, gene components and products includephotosynthesis, sugar metabolism, starch synthesis, starch degradation,nitrate and ammonia metabolism, amino acid biosynthesis, transport,protein biosynthesis, DNA replication, repair, lipid biosynthesis andbreakdown, protein biosynthesis, storage and breakdown, nucleotidetransport and metabolism, cell envelope biogenesis, membrane formation,mitochondrial and chloroplast biogenesis, transcription and mametabolism, vitamin biosynthesis, steroid and terpenoid biosynthesis,devise secondary metabolite synthesis, co-enzyme metabolism, flavonoidbiosynthesis and degradation, synthesis of waxes, glyoxylate metabolism,and hormone perception and response pathways.

Use of Plants that are Modified as Described Above

Altering leaf genes or gene products in a plant modifies one or more thefollowing plant traits, to make the plants more useful for specificpurposes in agriculture, horticulture and for the production of valuablemolecules. The useful plants have at least one of the followingcharacteristics: More seedling vigor; a higher yield of early leaves andtheir molecular constituents due to different number, size, weight,harvest index, composition including and amounts and types ofcarbohydrates, proteins, oils, waxes, etc., photosynthetic efficiency,e.g. reduced photorespiration, absorption of water and nutrients toenhance yields, including under stresses such as high light, herbicides,and heat, pathways to accumulate new valuable molecules; more optimalleaf shape and architecture in early seedling-enhancing photosynthesisand enhancing appeal in ornamental species including size, number, orpigment; a better overall plant architecture—enhancing photosynthesisand enhancing appeal in ornamental species; reduced negative effects ofhigh planting density, by altering leaf placement to be more verticalinstead of parallel to the ground; for instance better stress tolerance,including drought resistance, by decreasing water loss, and pathogenresistance; better overall yield and vigor—Plant yield of biomass and ofconstituent molecules and plant vigor are modulated to create benefitsby genetically changing the growth rate of seedling, coleoptileelongation, and young leaves.

To change any of the phenotype(s) above, activities of one or more ofthe early seedling phase genes or gene products are modulated in anorganism and the consequence evaluated by screening for the desiredtrait. Specifically, the gene, mRNA levels, or protein levels arealtered in a plant utilizing the procedures described herein and thephenotypes can be assayed. As an example, a plant can be transformedaccording to Bechtold and Pelletier (Methods. Mol. Biol. 82:259-266(1998)) with leaf gene constructs and/or screened for variants as inWinkler et al., Plant Physiol. 118: 743-50 (1998) and visually inspectedfor the desired phenotype and metabolically and/or functionally assayedfor altered levels of relevant molecules.

III.B.2.c. Use of Early Seedling Phase Genes, Gene Components andProducts to Modulate Biochemical Activities

Seedlings are complex and their structure, function and propertiesresult from the integration of many processes and biochemicalactivities. Some of these are known from the published literature andsome can be deduced from the genes and their products described in thisapplication. Early seedling phase genes, and gene components are usedsingly or in combination to modify these processes and biochemicalactivities and hence modify the phenotypic and trait characteristicsdescribed above. Examples of the processes and metabolic activities aregiven in the Table below. The resulting changes are measured accordingto the citations included in the Table.

BIOCHEMICAL OR METABOLIC ACTIVITIES AND/OR CITATIONS PROCESS PATHWAYSINCLUDING ASSAYS Metabolism - anabolic G. Farnesylation Pei et al.,Science 282: 287-290 and catabolic H. Cell (1998); Cutler et al.,Science 273: Wall 1239 (1996) Biosynthesis Goupil et al., J Exptl.Botany I. Nitrogen 49: 1855-62 (1998) Metabolism Walch-Liu et al., JExppt. Botany J. Secondary 51, 227-237 (2000) Metabolite Biosynthesisand Degradation Water Conservation And A. Production of Allen et al.,Plant Cell 11: 1785-1798 Resistance To Drought polyols (1999) And OtherRelated B. Regulation of Li et al., Science 287: 300-303 Stresses salt(2000) Transport Anion and concentration Burnett et al., J Exptl. Botany51: Cation Fluxes C. ABA 197-205 (2000) response(s) Raschke, In:Stomatal Function, (i) Ca2+ Zeiger et al. Eds., 253-279 (1987)Accumulation Lacombe et al., Plant Cell 12: 837-51 (a) K+ Fluxes (2000);(b) Na+ Fluxes Wang et al., Plant Physiol. 1. Receptor - 118: 1421-1429(1998); ligand binding Shi et al., Plant Cell 11: 2393-2406 2. Anion and(1999) Cation fluxes Gaymard et al., Cell 94: 647-655 (1998) Jonak etal., Proc. Natl. Acad. Sci. 93: 11274-79 (1996); Sheen, Proc. Natl.Acad. Sci. 95: 975-80 (1998); Allen et al., Plant Cell 11: 1785-98(1999) Carbon Fixation 3. Calvin Cycle Wingler et al., Philo Trans R Soe5. Photorespiration Lond B Biol Sci 355, 1517-1529 6. Oxygen (2000);evolution Palecanda et al., Plant Mol Biol 7. RuBisCO 46, 89-97 (2001);4. Chlorophyll Baker et al., J Exp Bot 52, 615-621 metabolism (2001)(ii) Chloroplast Chen et al., Acta Biochim Pol 41, Biogenesis 447-457(1999) and Imlau et al., PlantCell II, 309-322 Metabolism (1999) 5.Fatty Acid and Lipid Biosynthesis (iii) Glyoxylate metabolism (iv) SugarTransport (v) Starch Biosynthesis and Degradation Hormone Perception and(vi) Hormone Tieman et al., Plant J 26, 47-58 Growth Receptors (2001)and Hilpert et al., Plant J 26, 435-446 Downstream (2001) PathwaysWenzel et al., Plant Phys 124, for 813-822 (2000) (a) ethylene Denglerand Kang, Curr Opin (b) jasmonic acid Plant Biol 4, 50-56 (2001) (c)brassinosteroid Tantikanjana et al., Genes Dev 15, (d) gibberellin1577-1580 (2001) (e) Auxin (f) cytokinin Activation Of Specific KinasesAnd Phosphatases See Imbibition, Shoot Apical Meristem, Root and Leafsections for more details

Other biological activities that are modulated by the leaf genes andgene products are listed in the Reference tables. Assays for detectingsuch biological activities are described in the Protein Domain table,for example.

III.B.2.d. Use of Early Seedling Phase Genes, Gene Components andProducts to Modulate Transcription Levels

The expression of many genes is “up regulated” or down regulated” inplants because some genes and their products are integrated into complexnetworks that regulate transcription of many other genes. Some earlyseedling phase genes, gene components and products are therefore usefulfor modifying the transcription of other genes and hence complexphenotypes, as described above. Profiles of leaf gene activities aredescribed in the Table below with associated biological activities.“Up-regulated” profiles are those where the mRNA transcript levels arehigher in young seedlings as compared to the sterilized seeds.“Down-regulated” profiles represent higher transcript levels in theplantlet as compared to sterilized seed only.

III.B.3. Size and Stature Genes, Gene Components and Products

Great agronomic value can result from modulating the size of a plant asa whole or of any of its organs. For example, the green revolution cameabout as a result of creating dwarf wheat plants, which produced ahigher seed yield than taller plants because they could withstand higherlevels and inputs of fertilizer and water. Size and stature geneselucidated here are capable of modifying the growth of either anorganism as a whole or of localized organs or cells. Manipulation ofsuch genes, gene components and products can enhance many traits ofeconomic interest from increased seed and fruit size to increasedlodging resistance. Many kinds of genes control the height attained by aplant and the size of the organs. For genes additional to the ones inthis section other sections of the Application should be consulted.

III.B.3.a. Identification of Size and Stature Genes, Gene Components andProducts

Size and stature genes identified herein are defined as genes, genecomponents and products capable of modulating one or more processes ingrowth and development, to produce changes in size of one or moreorgans. Examples of such stature genes and gene products are shown inthe Reference, Sequence, Protein Group, Protein Group Matrix, Knock-in,Knock-out, MA-diff and MA-clust. The biochemical functions of theprotein products of many of these genes determined from comparisons withknown proteins are also given in the Reference tables.

Size and Stature Genes, Gene Components and Products Identified byPhenotypic Observations

Mutant plants exhibiting increased or decreased stature in comparison toparental wild-type plants were used to identify size and stature genes.In these experiments, size and stature genes were identified by either(1) the ectopic expression of a cDNA in a plant or (2) mutagenesis ofthe plant genome. The plants were then cultivated and stature genes wereidentified from plants that were smaller than the parental “wild-type”.The phenotypes and gene mutations associated with them are given inTables Examples of phenotypes, biochemical activities, or transcriptprofiles that are modulated using these genes are described above andbelow.

Use of Size and Stature Genes, Gene Components and Products to ModulatePhenotypes

Typically, these genes can cause or regulate cell division, rate andtime; and also cell size and shape. Many produce their effects viameristems. These genes can be divided into three classes. One class ofgenes acts during cytokinesis and/or karyokinesis, such as mitosisand/or meiosis. A second class is involved in cell growth; examplesinclude genes regulating metabolism and nutrient uptake pathways.Another class includes genes that control pathways that regulate orconstrain cell division and growth. Examples of these pathways includethose specifying hormone biosynthesis, hormone sensing and pathwaysactivated by hormones.

Size and stature genes and gene components are useful to selectivelyalter the size of organs and stems and so make plants specificallyimproved for agriculture, horticulture and other industries. There are ahuge number of utilities. For example, reductions in height of specificornamentals, crops and tree species can be beneficial, while increasingheight of others may be beneficial.

Increasing the length of the floral stems of cut flowers in some specieswould be useful, while increasing leaf size in others would beeconomically attractive. Enhancing the size of specific plant parts,such as seeds, to enhance yields by stimulating hormone (Brassinolide)synthesis specifically in these cells would be beneficial. Anotherapplication would be to stimulate early flowering by altering levels ofgibberellic acid in specific cells. Changes in organ size and biomassalso results in changes in the mass of constituent molecules. This makesthe utilities of size and stature genes useful for the production ofvaluable molecules in parts of plants, for extraction by the chemicaland pharmaceutical industries.

Examples of phenotypes that can be modulated by the genes and genecomponents include cell size, cell shape, cell division, rate anddirection, cell elongation, cell differentiation, stomata number, andtrichome number. The genes of the invention are useful to regulate thedevelopment and growth of roots (primary, lateral, root hairs, root cap,apical meristem, epidermis, cortex, and stele); stem (pholem, xylem,nodes, internodes, and shoot apical meristem); leaves (cauline, rosette,and petioles); flowers (receptacle, sepals, petals, and tepals,including color, shape, size, number, and petal drop, androecium,stamen, anther, pollen, sterility, size, shape, weight, color, filament,gynoecium, carpel, ovary, style, stigma, ovule, size, shape, and number,pedicel and peduncle, flowering time, and fertilization); seeds(placenta, embryo, cotyledon, endosperm, suspensor, and seed coat(testa)); and fruits (pericarp—thickness, texture, exocarp, mesocarp,and endocarp. Traits can be modulated with the genes and gene productsof this invention to affect the traits of a plant as a whole includearchitecture (such as branching, ornamental architecture, shadeavoidance, planting density effects, and wind resistance) and vigor(such as increased biomass and drought tolerance).

To regulate any of the phenotype(s) above, activities of one or more ofthe sizing genes or gene products are modulated in an organism andtested by screening for the desired trait. Specifically, the gene, mRNAlevels, or protein levels can be altered in a plant utilizing theprocedures described herein and the phenotypes can be assayed. As anexample, a plant can be transformed according to Bechtold and Pelletier(Methods. Mol. Biol. 82:259-266 (1998)) and/or screened for variants asin Winkler et al., (Plant Physiol. 118: 743-50 1998) and visuallyinspected for the desired phenotype or metabolically and/or functionallyassayed.

III.B.3.b. Use of Size and Stature Genes, Gene Components and Productsto Modulate Biochemical Activities

Many metabolic and developmental processes can be modulated by size andstature genes and gene components to achieve the phenotypiccharacteristics exemplified above. Some of these are listed below. Suchbiological activities can be measured according to the citationsincluded in the Table below:

BIOCHEMICAL OR METABOLIC ACTIVITIES CITATIONS INCLUDING PROCESS AND/ORPATHWAYS ASSAYS Growth and Development Gibberellic Acid BiosynthesisSwain SM, Tseng Ts, Gibberellic Acid Receptor and Olszewski NE. AlteredDownstream Pathways expression of spindly affects gibberellin responseand plant development. Plant Physiol 2001 July; 126(3): 1174-85 Hooley,R. Gibberellins: perception, transduction, and responses. Plant Mol.Biol. 1994 26: 1529-1555. Hooley, R. Gibberellins: perception,transduction, and responses. Plant Mol. Biol. 1994 26: 1529-1555.Perata, P, Matsukura, C, Vernieri, P, Yamaguchi, J, Sugar repression ofa gibberellin-dependent signaling pathway in barley embryos. Plant Cell1997 9: 2197-2208. Brassinolide Biosynthesis Noguchi T, Fujioka S, ChoeS, Brassinolide Receptors, Takatsuto S, Tax FE, Yoshida S, Degradationof Brassinolide Feldmann KA. Biosynthetic Pathways affected by pathwaysof brassinolide in Brassinolide Arabidopsis. Plant Physiol 2000September; 124(1): 201-9 Wang ZY, Seto H, Fujioka S, Yoshida S, Chory J.BRI1 is a critical component of a plasma-membrane receptor for plantsteroids. Nature 2001 Mar 15; 410(6826): 380-3 Neff MM, Nguyen SM,Malancharuvil EJ, Fujioka S, Noguchi T, Seto H, Tsubuki M, Honda T,Takatsuto S, Yoshida S, Chory J. BAS1: A gene regulating brassinosteroidlevels and light responsiveness in Arabidopsis. Proc Natl Acad Sci USA1999 Dec 21; 96(26): 15316-23 Kang JG, Yun J, Kim DH, Chung KS, FujiokaS, Kim JI, Dae HW, Yoshida S, Takatsuto S, Song PS, Park CM. Light andbrassinosteroid signals are integrated via a dark-induced small Gprotein in etiolated seedling growth. Cell 2001 Jun 1; 105(5): 625-36Cytokinin biosynthesis Mok DW, Mok MC. Cytokinin Cytokinin receptormetabolism and action. Annu Degradation of Cytokinin Rev Plant PhysiolPlant Mol Pathways affected by Cytokinin Biol 2001; 52: 89-118Schmulling T. CREam of cytokinin signalling: receptor identified. TrendsPlant Sci 2001 July; 6(7): 281-4 Mok DW, Mok MC. Cytokinin metabolismand action. Annu Rev Plant Physiol Plant Mol Biol 2001; 52: 89-118Seyedi M, Selstam E, Timko MP, Sundqvist C. The cytokinin2-isopentenyladenine causes partial reversion to skotomorphogenesis andinduces formation of prolamellar bodies and protochlorophyllide657 inthe lip1 mutant of pea. Physiol Plant 2001 June; 112(2): 261-272 AuxinBiosynthesis Zhao Y, Christensen SK, Auxin Receptor Fankhauser C,Cashman JR, Auxin Degradation Cohen JD, Weigel D, Chory J. Pathwaysaffected by Auxins A role for flavin Auxin transport monooxygenase-likeenzymes in Auxin biosynthesis. Science 2001 Jan 12; 291(5502): 306-9Abel S, Ballas N, Wong LM, Theologis A. DNA elements responsive toAuxin. Bioessays 1996 August; 18(8): 647-54 del Pozo JC, Estelle M.Function of the ubiquitin- proteosome pathway in Auxin response. TrendsPlant Sci 1999 March; 4(3): 107-112. Rahman A, Amakawa T, Goto N,Tsurumi S. Auxin is a positive regulator for ethylene- mediated responsein the growth of Arabidopsis roots. Plant Cell Physiol 2001 March;42(3): 301-7 Zhao Y, Christensen SK, Fankhauser C, Cashman JR, Cohen JD,Weigel D, Chory J. A role for flavin monooxygenase-like enzymes in Auxinbiosynthesis. Science 2001 Jan 12; 291(5502): 306-9 Abel S, Ballas N,Wong LM, Theologis A. DNA elements responsive to Auxin. Bioessays 1996August; 18(8): 647-54 del Pozo JC, Estelle M. Function of the ubiquitin-proteosome pathway in Auxin response. Trends Plant Sci 1999 March; 4(3):107-112. Rahman A, Amakawa T, Goto N, Tsurumi S. Auxin is a positiveregulator for ethylene- mediated response in the growth of Arabidopsisroots. Plant Cell Physiol 2001 March; 42(3): 301-7 Gil P, Dewey E, FrimlJ, Zhao Y, Snowden KC, Putterill J, Palme K, Estelle M, Chory J. BIG: acalossin-like protein required for polar Auxin transport in Arabidopsis.Genes Dev. 2001 Aug 1; 15(15): 1985-97 Estelle M., Polar Auxintransport. New support for an old model. Plant Cell 1998 November;10(11): 1775-8 Cell wall growth Cosgrove DJ., Loosening of plant cellwalls by expansins. Nature 2000 Sep 21; 407(6802): 321-6

Other biological activities that are modulated by the stature genes andgene products are listed in the Reference tables. Assays for detectingsuch biological activities are described in the Protein Domain table,for example.

Changes in the size, vigor, or yield of a plant are the result ofmodulation of the activities of one or more of these many size andstature genes and gene products. While size and stature polynucleotidesand gene products can act alone, combinations of these polynucleotidesand also with others that also affect growth and development areespecially useful.

Use of Promoters of “Size and Stature” Genes

Promoters of “size and stature” genes are useful for controlling thetranscription of any desired polynucleotides, both plant and non-plant.They can be discovered from the “size and stature” genes in theReference Tables, and their patterns of activity from the MA Tables.When operably linked to any polynucleotide encoding a protein, andinserted into a plant, the protein will be synthesized in those cells inwhich the promoter is active. Many “size and stature” genes willfunction in meristems, so the promoters will be useful for expressingproteins in meristems. The promoters can be used to cause loss of, aswell as synthesis of, specific proteins via antisense and sensesuppression approaches.

III.B.4. Shoot-Apical Meristem Genes, Gene Components and Products

New organs, stems, leaves, branches and inflorescences develop from thestem apical meristem (SAM). The growth structure and architecture of theplant therefore depends on the behavior of SAMs. Shoot apical meristems(SAMs) are comprised of a number of morphologically undifferentiated,dividing cells located at the tips of shoots. SAM genes elucidated hereare capable of modifying the activity of SAMs and thereby many traits ofeconomic interest from ornamental leaf shape to organ number toresponses to plant density.

III.B.4.a. Identification of Sam Genes, Gene Components and Products

SAM genes identified herein are defined as genes, gene components andproducts capable of modulating one or more processes or functions ofSAMs as described below. Regulation of SAM genes and gene products areuseful to control many plant traits including architecture, yield andvigor. Examples of such SAM genes and gene products are shown in theReference, Sequence, Protein Group, Protein Group Matrix, phenotype andMA-diff Tables. The functions of many of the protein products of thesegenes are also given in the Reference tables.

Sam Genes, Gene Components and Products Identified by PhenotypicObservations

SAM genes were discovered and characterized from a much larger set ofgenes by experiments designed to find genes that cause phenotypicchanges in leaf morphology, such as cotyledon or leaf fusion. In theseexperiments, SAM genes were identified by either (1) ectopic expressionof a cDNA in a plant or (2) mutagenesis of the plant genome. The plantswere then cultivated and one or more of the following phenotypes, whichvaried from the parental “wild-type”, was observed:

I. Cotyledon

-   -   Fused

II. Leaves

-   -   Fused    -   Leaf placement on stems

III. Branching

-   -   Number

IV. Flowers

-   -   Petals fused    -   Altered bolting    -   Early bolting    -   Late bolting    -   Strong bolting    -   Weak bolting    -   Abnormal branching

For more experimental detail see the Example section below. The genesidentified by these results of the phenotypes that are shown in Knock-inand Knock-out Tables.

Sam Genes, Gene Components and Products Identified by DifferentialExpression

SAM genes were also identified in experiments designed to find geneswhose mRNA products are associated specifically or preferentially withSAMs. The concentration of mRNA products in the arabidopsis plant withthe SHOOTMERISTEMLESS (STM) gene knocked-out was measured relative tothe concentration in the parental, non-mutant plant. The Arabidopsis STMgene is required for embryonic SAM formation. The STM gene encodes aKnotted1 (Kn1) type of homeodomain protein. Homeodomain proteinsregulate transcription of many genes in many species and have been shownto play a role in the regulation of translation as well. Seedlingshomozygous for recessive loss-of-function alleles germinate with roots,a hypocotyl, and cotyledons, but no SAM is formed. The MA_diff Table(s)reports the transcript levels of the experiment (see EXPT ID: 108478,108479, 108480, 108481, 108598, 108535, 108536, 108435). For transcriptsthat had higher levels in the samples than the control, a “+” is shown.A “−” is shown for when transcript levels were reduced in root tips ascompared to the control. For more experimental detail see the Examplesection below.

Meristem genes are those sequences that showed differential expressionas compared to controls, namely those sequences identified in theMA_diff tables with a “+” or “−” indication.

Meristem Genes Identified by Cluster Analyses of Differential Expression

Meristem Genes Identified by Correlation to Genes that areDifferentially Expressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of Meristem genes is any group in the MA_clust thatcomprises a cDNA ID that also appears in Expt ID 108478, 108479, 108480,108481, 108598, 108535, 108536, 108435 of the MA_diff table(s).

Meristem Genes Identified by Correlation to Genes that CausePhysiological Consequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of Meristemgenes. A group in the MA_clust is considered a Meristem pathway ornetwork if the group comprises a cDNA ID that also appears in Knock-inor Knock-out tables that causes one or more of the phenotypes describedin section above.

Meristem Genes Identified by Amino Acid Sequence Similarity

Meristem genes from other plant species typically encode polypeptidesthat share amino acid similarity to the sequences encoded by corn andArabidopsis Meristem genes. Groups of Meristem genes are identified inthe Protein Group table. In this table, any protein group that comprisesa peptide ID that corresponds to a cDNA ID member of a Meristem pathwayor network is a group of proteins that also exhibits Meristemfunctions/utilities.

Examples of phenotypes, biochemical activities, and transcriptionprofiles that can be modulated by SAM genes and gene products aredescribed above and below.

III.B.4.b. Use of Sam Genes Gene Components and Products to ModulatePhenotypes

With the SAM genes and gene products of the invention, Applicantsprovide the means to modulate one or more of the following types ofSAMs:

1. Embryonic meristem

2. Vegetative lateral SAMs

3. Inflorescence lateral SAMs

4. Floral meristems

5. Adventitious SAM

The SAM genes of the instant invention are useful for modulating one ormore processes of SAM structure and/or function including (I) cell sizeand division; (II) cell differentiation and organ primordia.

I. Cell Size and Division

A. Cell Properties

SAM genes and gene products can be used to modulate changes in cellsize, cell division, rate and direction, and cell division symmetry.

A key attribute of the SAM is its capacity for self-renewal. Theself-renewing initial cell population resides in the central zone of theSAM. A small number of slowly dividing initial cells (typically 2 to 4per layer) act as a self-replenishing population, whereas some of theirdescendants, pushed out onto the flanks of the SAM, differentiate intoleaves. Other descendants, displaced below the SAM, differentiate intostem. The immediate descendants of the initial cells divide further,amplifying the cell population before being incorporated into leaf orstem primordia.

The genes and gene components of this invention are useful formodulating any one or all of these cell division processes generally, asin timing and rate, for example. In addition, the polynucleotides andpolypeptides of the invention can control the response of theseprocesses to the internal plant programs associated with embryogenesis,hormone responses like cytokinin (inhibitory for root development, seesection on cytokinin-responsive genes), coordination of growth anddevelopment with that of other plant organs (such as leaves, flowers,seeds, and fruits.

SAM genes can also be used to control the response of these processes tochanges in the environment, including heat, cold, drought, high lightand nutrition.

B. Sam Cell Patterns and Organization

Although SAMs appear as small regions of morphological undifferentiateddividing cells, a group of morphologically undifferentiated dividingcells does not necessarily constitute a SAM. Rather, evidence indicatesthat SAMs are highly organized or patterned regions of the plant inwhich many important events in early organogenesis occur. Thus, the term“SAM” is used to denote a highly organized structure and site of patternformation. The invention also permits engineering of specific as well asoverall features of SAM architecture including zones (central,peripheral, and rib), layers (l1, l2, and l3) and symmetry.

II Cell Differentiation and Organ Primordia

The apical meristem in many species first undergoes a vegetative phasewhereby cells set aside from the apex become leaf primordia with anaxillary vegetative meristem. Upon floral induction, the apical meristemis converted to an inflorescence meristem. The inflorescence meristemarises in the axils of modified leaves and is indeterminate, producingwhorls or rings of floral organ primordia. In species which produceterminal flowers, the apical meristem is determinate and eventuallyadopts a third identity, that of a floral meristem. Examples of theplant properties that the genes and gene products of the invention canbe used to modulate include indeterminacy (inhibiting or increasingdifferentiation and enhancing plant growth and yield), symmetry(symmetry of organs developed, and symmetry of arrangement of organs,such as leaves, petals, flowers, etc.), leaf fate and timing internodelength modulation, such as longer internodes to increase shade avoidanceand shorter internodes to favor leaf development), and floral fate andtiming of flowering.

Uses of Plants Modified as Described Above Using Sam Genes, GeneComponents and Products

Because SAMs determine the architecture of the plant, modified plantswill be useful in many agricultural, horticultural, forestry and otherindustrial sectors. Plants with a different shape, numbers of flowersand seed and fruits will have altered yields of plant parts. Forexample, plants with more branches can produce more flowers, seed orfruits. Trees without lateral branches will produce long lengths ofclean timber. Plants with greater yields of specific plant parts will beuseful sources of constituent chemicals. Such plants will have, forexample, more prolific leaf development, better optimized stem and shootdevelopment, adventitious shoots, more flowers, seeds, and fruits,enhanced vigor (including growth rate of whole plant, including height,flowering time, etc., seedling, coleoptile elongation, young leaves,flowers, seeds, and fruit. higher yields based on biomass (fresh and dryweight during any time in plant life, including maturation andsenescence), number of flowers, seed yield (number, size, weight,harvest index, content and composition, e.g. amino acid, jasmonate, oil,protein and starch) and fruit yield (number, size, weight, harvestindex, content and composition, e.g. amino acid, jasmonate, oil, proteinand starch).

To regulate any of the phenotype(s) above, activities of one or more ofthe SAM genes or gene products can be modulated and tested by screeningfor the desired trait. Specifically, the gene, mRNA levels, or proteinlevels can be altered in a plant utilizing the procedures describedherein and the phenotypes can be assayed. As an example, a plant can betransformed according to Bechtold and Pelletier (1998, Methods. Mol.Biol. 82:259-266) and/or screened for variants as in Winkler et al.(1998) Plant Physiol 118: 743-50 and visually inspected for the desiredphenotype or metabolically and/or functionally assayed according toDolan et al. (1993, Development 119: 71-84), Dolan et al. (1997,Development 124: 1789-98), Crawford and Glass (1998, Trends PlantScience 3: 389-95), Wang et al. (1998, PNAS USA 95: 15134-39), Gaxiolaet al. (1998, PNAS USA 95: 4046-50), Apse et al. (1999, Science 285:1256-58), Fisher and Long (1992, Nature 357: 655-60), Schneider et al.(1998, Genes Devel 12: 2013-21) and Hirsch (1999, Curr Opin Plant Biol.2: 320-326).

III.B.4.c. Use of Sam Genes and Gene Components to Modulate BiochemicalActivities

SAM genes and gene components are useful for modulating biochemical ormetabolic activities and/or pathways such as those noted below. Suchbiological activities can be measured according to the citationsincluded in the Table below:

BIOCHEMICAL OR METABOLIC ACTIVITIES CITATIONS INCLUDING PROCESS AND/ORPATHWAYS ASSAYS Growth, Differentiation Leaf shape and inflorescence andChuck, G. et al., 1996 Plant Cell And Development flower morphologysystems 8: 1227-1289. Activities of SAM Schneeberger et al., 1998transcriptional regulatory Development 125: 2857-2865. proteins.Meristem size and organ number Kayes, J. M. and Clark, S. E.determinants 1998 Development 125: 3843-3851. Regulated by ReceptorKinases Jeong, S. et al., 1999 Plant Receptor kinase location and Cell11: 1925-1934. activity. Meristem proliferation activities Tantikanjana,T. Genes and Development. Jun. 15, 2001. 15(12): 1577-1588. Internodeelongation Hormone signaling pathways Yamamuro, C. et al., 2000 PlantCell. 12: 1591-1605. Hormone Levels of growth hormones Kusaba, S. et al;1998 Plant Perception including gibberellic acid, Auxin Physiology116(2): 471-476. and cytokinin. Gibberellic acid biosynthesis Modulationof GA perception GA biosynthetic enzyme GA-20 and function can beassayed as oxidase is a required step in GA described in Sakamoto, T. etal. biosynthesis. GA-20 oxidase is 2001 Genes and Development Regulatedby some SAM gene 15: 581-590. products. Over expression of SAM genesSakamoto, T. et al. 2001. can lead to reduced internode Genes andDevelopment 15: elongation, reduced cell 581-590. elongation and reducedcell expansion. Cytokinin Receptor activity Inoue, T. et al., Nature409: 1060-1063. SAM gene products can affect Sieberer, T. et al., 2000Current the activity of Auxin dependent Biology 10: 1595-1598.postranscriptional gene protein del Pozo, J. C.; Estelle, M. expression.PNAS (USA) 1999. 96(26): 15342-15347. SAM gene products can affectTantikanjana, T. Genes and Auxin Perception/metabolism in Development.Jun. 15, 2001. the meristem to produce useful 15(12): 1577-1588. changesin plant architecture. Leaf senescence SAM gene products can increaseOri, N. et al; Plant Cell. June, and decrease leaf senescence 1999.11(6): 1073-1080. rate. This can be done by modulating cytokinin hormonelevels. Cytokinin effect on cell division Beemster, Gerrit T. S.;Baskin, and expansion. Tobias I. 2000 Plant Physiology 124: 1718-1727.Adventitious shoot Alter growth hormone status. Kusaba, S. et al; 1998Plant formation Physiology 116(2): 471-476 Ectopic expression of SAMChuck, G. 1996 Plant Cell 8: genes in leaf or other non SAM 1227-1289.organs or tissue can produce shoots Pathways comprising isopentenyltransferase (ipt)

Other biological activities that can be modulated by the SAM genes andgene products are listed in the Reference tables. Assays for detectingsuch biological activities are described in the Protein Domain table.

III.B.4.d. Use of Sam Genes, Gene Components and Products to ModulateTranscription Levels of Other Genes

The expression of many genes is “upregulated” or “downregulated” in theSAM mutants because some of the SAM genes are integrated into complexnetworks that regulate the transcription of many other genes. Some SAMgenes and gene components are therefore useful for modifying thetranscription of other genes and hence complex phenotypes as describedabove. Profiles of genes altered by SAM mutations and genes aredescribed in the Table below with associated biological activities.“Up-regulated” profiles are for genes whose mRNA levels are higher inthe stm plants as compared to parental wild-type plants; and vice-versafor “down-regulated” profiles.

PHYSIOLOGICAL EXAMPLES OF TYPE OF GENES CONSEQUENCES OF BIOCHEMICALWHOSE MODIFYING SAM ACTIVITIES WHOSE TRANSCRIPT TRANSCRIPTS ARE GENEPRODUCT TRANSCRIPTS ARE LEVELS CHANGED LEVELS CHANGED Up Regulated Genesrepressed by Altered Transporters Transcripts SAMs directly orAuxin/cytokinin Metabolic Enzymes indirectly hormone ratio and CellMembrane perception. Structure Increased/decreased Kinases,Phosphatases, cell expansion - G-Proteins promoting effects ofTranscription brassinosteroids and Activators/Repressors gibberellicacids, due Transcription to altered levels of coactivators/corepressorsbiosynthetic pathway Chromatin Structure enzymes and or the And/OrLocalized DNA amount of functional Topology Proteins hormone receptor.Cell Wall Proteins Increased or Translational decreased rate of cellactivators/repressors division. Cell wall proteins Altered planes ofcell involved in cell rigidity division e.g. extensin, glycine Increasedor rich proteins. decreased rate and Cell cycle regulatory extent ofcell proteins such as cyclins expansion. and cyclin dependent Increasedor protein kinases (CDKs). decreased rigidity of cell ways.Down-Regulated Genes involved in SAM Altered pattern of Auxintransporter Transcripts cells and genes whose organs immerging proteinsexpression is induced by from the meristem Auxin receptor proteins SAMsIncreased or Cytokinin receptor decreased the number proteins of cellspartitioned Gibberellic acid receptor into a lateral organ. proteinsAltered apical Brassinolide receptor dominance due to proteinssuppression of lateral Hormone biosynthesis bud growth. proteins Alteredapical Hormone degradation dominance due to proteins releasing ofaxillary Hormone conjugation meristems from proteins repression.Ubiquitin conjugating Increased/or enzymes. decreased productionReceptor kinase signal of adventitious transduction meristems. Increasedpotential to form somatic embryos. Altered cell signaling pathwaysAltered hormone levels

SAM genes and gene products can be modulated alone or in combination asdescribed in the introduction. Of particular interest are combination ofthese genes and gene products with those that modulate hormoneresponsive pathways. Hormone responsive genes and gene products aredescribed in more detail in the sections below.

Use of Sam Gene Promoters to Modify SAMs

Promoters of SAM genes, as described in the Reference tables, forexample, can be used to modulate transcription of coding sequences inSAM cells to influence growth, differentiation or patterning ofdevelopment or any of the phenotypes or biological activities above. Forexample, any desired sequence can be transcribed in similar temporal,tissue, or environmentally specific patterns as a SAM gene when thedesired sequence is operably linked to the promoter of the SAM gene.

A specific instance is linking of a SAM gene promoter normally active infloral meristem primordia, to a phytotoxic protein coding sequence toinhibit apical meristem switching into an inflorescence and/or floralmeristem, thereby preventing flowering.

SAM gene promoters can also be used to induce transcription of antisenseRNA copies of a gene or an RNA variant to achieve reduced synthesis of aspecific protein in specific SAM cells. This provides an alternative wayto the example above, to prevent flowering.

EXAMPLES OF PHYSIOLOGICAL BIOCHEMICAL TYPE OF GENES CONSEQUENCESACTIVITIES OF WHOSE OF MODIFYING GENE PRODUCTS TRANSCRIPT TRANSCRIPTSGENE PRODUCT WITH MODIFIED LEVELS ARE CHANGED LEVELS LEVELS Up regulatedGenes involved in Leaf cells Transcription Transcripts leaf, stem androot proliferate and factors, signal cell differentiation,differentiate; transduction cell division, cell Leaf structuresproteins, kinase expansion form and expand and phosphatases Genesinvolved in Chromatin positive regulation of remodeling root, stem andleaf Hormone genes biosynthesis Repressors of root enzymes and otherorgan cell Receptors types e.g. flowers Genes involved in PhotosynthesisLight harvesting photosynthesis and plastid coupled to ATPdifferentiation production Calvin cycle Chlorophyll activatedbiosynthesis Chloroplast Ribulose biogenesis and Bisphosphate plastidcarboxylase differentiation Chloroplast activated membranes synthesisChloroplast ribosome biogenesis Other genes involved in Starch Starchsynthase metabolism biosynthesis Nitrate reductase Lipid Terpenoidbiosynthesis biosynthesis Nitrogen Transcription metabolism - factorsNO3 reduced and Transporters amino acids made Kinases SecondaryPhosphatases and metabolites signal produced transduction proteinChromatin structure modulators Down regulated Genes involved in Leafgenes Transcription genes negative regulation activated and leaf factorsof root, stem and leaf functions induced Signal genes Other organs nottransduction Genes involved in induced proteins - kinases other organse.g. Leaf, stem and and phosphatases flowers root metabolic Metabolicpathways induced enzymes Chromatin remodeling proteins

While early seedling phase polynucleotides and gene products are usedsingly, combinations of these polynucleotides are often better tooptimize new growth and development patterns. Useful combinationsinclude different leaf polynucleotides and/or gene products with ahormone responsive polynucleotide. These combinations are useful becauseof the interactions that exist between hormone-regulated pathways,nutritional pathways and development.

Use of Early Seedling Phase Gene Promoters

Promoters of early seedling phase genes are useful for transcription ofdesired polynucleotides, both plant and non-plant. If the gene isexpressed only in the post-germination seedling, or in certain kinds ofleaf cells, the promoter is used to drive the synthesis of proteinsspecifically in those cells. For example, extra copies of carbohydratetransporter cDNAs operably linked to a early seedling phase genepromoter and inserted into a plant increase the “sink” strength ofleaves. Similarly, early seedling phase promoters are used to drivetranscription of metabolic enzymes that alter the oil, starch, protein,or fiber contents of the seedling. Alternatively, the promoters directexpression of non-plant genes that can, for instance, confer resistanceto specific pathogen. Additionally the promoters are used to synthesizean antisense mRNA copy of a gene to inactivate the normal geneexpression into protein. The promoters are used to drive synthesis ofsense RNAs to inactivate protein production via RNA interference.

III.B.5. Vegetative-Phase Specific Responsive Genes, Gene Components andProducts

Often growth and yield are limited by the ability of a plant to toleratestress conditions, including water loss. To combat such conditions,plant cells deploy a battery of responses that are controlled by a phaseshift, from so called juvenile to adult. These changes at distinct timesinvolve, for example, cotyledons and leaves, guard cells in stomata, andbiochemical activities involved with sugar and nitrogen metabolism.These responses depend on the functioning of an internal clock, thatbecomes entrained to plant development, and a series of downstreamsignaling events leading to transcription-independent andtranscription-dependent stress responses. These responses involvechanges in gene expression.

Manipulation of the activation of one or more genes controlling thephase changes is useful to modulate the biological processes and/orphenotypes listed below. Phase responsive genes and gene products canact alone or in combination. Useful combinations include phaseresponsive genes and/or gene products with similar transcriptionprofiles, similar biological activities, or members of the same orfunctionally related biochemical pathways. Whole pathways or segments ofpathways are controlled by transcription factor proteins and proteinscontrolling the activity of signal transduction pathways. Therefore,manipulation of such protein levels is especially useful for alteringphenotypes and biochemical activities of plants.

Phase responsive genes and gene products can function to either increaseor dampen the above phenotypes or activities. Characterization of phaseresponsive genes was carried out using microarray technology. Microarraytechnology allows monitoring of gene expression levels for thousands ofgenes in a single experiment. This is achieved by hybridizing labeledfluorescent cDNA pools to glass slides that contain spots of DNA (Schenaet al. (1995) Science 270: 467-70). The US Arabidopsis FunctionalGenomics Consortium (AFGC) has recently made public the results fromsuch microarray experiments conducted with AFGC chips containing about10,000 non-redundant ESTs, selected from about 37,000 randomly sequencedESTs generated from mRNA of different tissues and developmental stages.

The sequences of the ESTs showing at least two-fold increases ordecreases in a mutant of Arabidopsis thaliana, squint, that appears notto undergo phase changes and appears adult-like throughout its growthcycle, compared with wild type were identified, compared to the Ceresfull length cDNA and genomic sequence databanks, and equivalent. Ceresclones identified. The MA_diff tables reports the results of thisanalysis, indicating those Ceres clones which are up or down regulatedover controls, thereby indicating the Ceres clones which represent phaseresponsive genes. The MA_diff Table(s) reports the transcript levels ofthe experiment (see EXPT ID: Sqn (relating to SMD 7133, SMD 7137)). Fortranscripts that had higher levels in the samples than the control, a“+” is shown. A “−” is shown for when transcript levels were reduced inroot tips as compared to the control. For more experimental detail seethe Example section below.

Phase responsive genes are those sequences that showed differentialexpression as compared to controls, namely those sequences identified inthe MA_diff tables with a “+” or “−” indication.

Phase Responsive Genes Identified by Cluster Analyses of DifferentialExpression

Phase Responsive Genes Identified by Correlation to Genes that areDifferentially Expressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of phase responsive genes is any group in theMA_clust that comprises a cDNA ID that also appears in Expt ID Sqn(relating to SMD 7133, SMD 7137) of the MA_diff table(s).

Phase Responsive Genes Identified by Correlation to Genes that CausePhysiological Consequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of phaseresponsive genes. A group in the MA_clust is considered a phaseresponsive pathway or network if the group comprises a cDNA ID that alsoappears in Knock-in or Knock-out tables that causes one or more of thephenotypes described in section above.

Phase Responsive Genes Identified by Amino Acid Sequence Similarity

Phase responsive genes from other plant species typically encodepolypeptides that share amino acid similarity to the sequences encodedby corn and Arabidopsis phase responsive genes. Groups of phaseresponsive genes are identified in the Protein Grouping table. In thistable, any protein group that comprises a peptide ID that corresponds toa cDNA ID member of a phase responsive pathway or network is a group ofproteins that also exhibits Phase responsive functions/utilities.

Further, promoters of phase responsive genes, as described in Referencetables, for example, are useful to modulate transcription that isinduced by phase or any of the following phenotypes or biologicalactivities below. Further, any desired sequence can be transcribed insimilar temporal, tissue, or environmentally specific patterns as thephase responsive genes when the desired sequence is operably linked to apromoter of a phase responsive gene.

III.B.5.a. Use of Phase Responsive Genes to Modulate

PhenotypesPhase responsive genes and gene products are useful to ormodulate one or more phenotype including timing phenotypes, dormancy,germination, cotyledon opening, first leaves, juvenile to adulttransition, bolting, flowering, pollination, fertilization, seeddevelopment, seed set, fruit drop, senescence, epinasty, biomass, freshand dry weight during any time in plant life, such as maturation, numberof flowers, seeds, branches, and/or leaves, seed yield, includingnumber, size, weight, and/or harvest index, fruit yield, includingnumber, size, weight, and/or harvest index, plant development, time tofruit maturity, cell wall strengthening and reinforcement, stresstolerance, drought tolerance, flooding tolerance, and UV tolerance.

To regulate any of the phenotype(s) above, activities of one or more ofthe phase responsive genes or gene products can be modulated and theplants can be tested by screening for the desired trait. Specifically,the gene, mRNA levels, or protein levels can be altered in a plantutilizing the procedures described herein and the phenotypes can bescreened for variants as in Anderson et al. (1997) Plant Cell 9:1727-1743; Heintzen et al. (1997) Proc. Natl. Acad. Sci. USA 94:8515-20; Schaffer et al. (1998) Cell 93:1219-1229; Somers et al. (1998)Development 125: 485-494; Somers et al. (1998) Science 282: 1488-1490;Wang and Tobin (1998) Cell 93: 1207-1217; Zhong et al. (1998) Plant Cell10: 2005-2017; Sugano et al. (1998) Proc. Natl. Acad. Sci. USA 95:11020-11025; Dowson-Day and Millar (1999) Plant J 17: 63-71; Green andTobin (1999) Proc. Natl. Acad. Sci. USA 96: 4176-419; Staiger and Apel(1999) Mol. Gen. Genet. 261: 811-819; Strayer and Kay (1999) Curr. Opin.Plant Biol. 2:114-120; Strayer et. al. (2000) Science 289:768-771; Krepset al. (2000) J Biol Rhythms (2000) 15:208-217; Nelson et al. (2000)Cell 101:331-340; Somers et al. (2000) Cell 101:319-329.

III.B.5.b. Use of Phase Responsive Genes to Modulate BiochemicalActivities

The activities of one or more of the phase responsive genes can bemodulated to change biochemical or metabolic activities and/or pathwayssuch as those noted below. Such biological activities are documented andcan be measured according to the citations above and included in thetable below:

Biochemical Or Metabolic Process Activities And/Or Pathways Citationsincluding assays Germination And Cold, Light And Water Bognar et al.(1999) Proc. Natl. Acad. Seedling Modulated Signal Transduction Sci. USA96: 14652-14657; Sugano et Development Pathways, Receptors, Kinases, al(1999) Proc. Natl. Acad. Sci. USA PAS Domain Proteins 96: 12362-12366;Dowson-Day and Millar (1999) Plant J 17: 63-71; Somers et al. (2000)Cell 101: 319-329; Zhong et al. (1998) Plant Cell 10: 2005-2017 GrowthCold And Light Modulated Nelson et al. (2000) Cell 101: 331-340;Transitions And Signal Transduction Pathways, Fowler et al. (1999) EMBOJ. Flowering Receptors, Kinases, PAS 18: 4679-4688 Domain Protiens TuberFormation Cold And Light Modulated Yanovsky et al. (2000) Plant J. 23:Signal Transduction Pathways 223-232 METABOLISM Lipid MetabolismMembrane Lipid Synthesis Bradley and Reddy (1997) J. Including Omega-3Fatty Acid Bacteriol. 179: 4407-4410; Martin, M Desaturase, Lipases,Lipid et al. 1999 Europe J. Biochem 262: Transfer Proteins 283-290 SugarGlycosylhydrolases, Liu et al. (1996) Plant Physiol. MetabolismGlycosyltransferases, 112: 43-51; Millar and Kay (1996) Amylases,Sucrose Synthase, Proc Natl Acad Sci USA 93: 15491-15496; CAB, Rubisco,Light Signal Wang et al. (1997) Plant Cell Transduction 9: 491-507;Shinohara et al (1999) J. Biol. Chem. 273: 446-452 NitrogenAminotransferases, Arginase, Bradley and Reddy (1997) J. MetabolismProteases And Vegetative Bacteriol. 179: 4407-4410 Storage Proteins,Aromatic Amino Acid Synthesis Photorespiration Mitochondrial,Chloroplast And Zhong and McClung (1996) Mol. Gen. PeroxisomalPhotorespiratory Genet. 251: 196-203; McClung (1997) Enzymes, SerineFree. Radic. Biol. Med. 23: 489-496; Hydroxymethyl Transferases, McClunget al. (2000) Plant Physiol. Catalase 123: 381-392 Responses ToExpression Of Genes Involved McClung (1997) Free Radic Biol MedEnvironmental In Responses To Drought, Salt, 23: 489-496; Shi et al.(2000) Proc. Stress UV Natl. Acad. Sci. USA 97: 6896-6901

Other biological activities that can be modulated by the phaseresponsive genes and their products are listed in the Reference tables.Assays for detecting such biological activities are described in theProtein Domain table.

Phase responsive genes are characteristically differentially transcribedin response to maturity of the cell, organ or tissue which depends on atiming mechanism, which is internal to an organism or cell. TheIntensity Table reports the changes in transcript levels of variousphase responsive genes in a plant.

The data from this experiment reveal a number of types of phaseresponsive genes and gene products. Profiles of some classes of phaseresponsive genes are shown in the table below with examples of whichassociated biological activities are modulated when the activities ofone or more such genes vary in plants.

Transcript Physiological Examples Of Biochemical Levels Type Of GenesConsequences Activity Up Regulated Responders To Adult phase MetabolicEnzymes Transcripts mutation that confers adoption Change In Cell adultlike phase Metabolisms Membrane Structure Genes induced in Affected Byphase And Potential adult-like phase change Kinases And Synthesis OfPhosphatases Secondary Transcription Metabolites Activators And/OrProteins Change In Chromatin Modulation Of Structure And/Or PhaseResponse Localized DNA Transduction Topology Pathways Specific GeneTranscription Initiation Down- Responders To Negative TranscriptionFactors Regulated mutation that confers Regulation of Change In ProteinTranscripts adult phase adult phase Structure By Genes repressed inpathways Phosphorylation adult-like phase Changes In (Kinases) Or GenesWith Pathways And Dephosphoryaltion Discontinued Processes(Phosphatases) Expression Or Operating In Cells Change In ChromatinUnstable mRNA in Changes In Structure And/Or adult-like phase MetabolicDNA Topology pathways other Stability Factors For than phase ProteinSynthesis And specific pathways Degradation Metabolic Enzymes

Use of Promoters of Phase Responsive Genes

Promoters of phase responsive genes are useful for transcription of anydesired polynucleotide or plant or non-plant origin. Further, anydesired sequence can be transcribed in a similar temporal, tissue, orenvironmentally specific patterns as the phase responsive genes wherethe desired sequence is operably linked to a promoter of a phaseresponsive gene. The protein product of such a polynucleotide is usuallysynthesized in the same cells, in response to the same stimuli as theprotein product of the gene from which the promoter was derived. Suchpromoter are also useful to produce antisense mRNAs to down-regulate theproduct of proteins, or to produce sense mRNAs to down-regulate mRNAsvia sense suppression.

III.C. Hormone Responsive Genes, Gene Components and Products

III.C.1. Abscissic Acid Responsive Genes, Gene Components and Products

Plant hormones are naturally occurring substances, effective in verysmall amounts, which act as signals to stimulate or inhibit growth orregulate developmental processes in plants. Abscisic acid (ABA) is aubiquitous hormone in vascular plants that has been detected in everymajor organ or living tissue from the root to the apical bud. The majorphysiological responses affected by ABA are dormancy, stress stomatalclosure, water uptake, abscission and senescence. In contrast to Auxins,cytokinins and gibberellins, which are principally growth promoters, ABAprimarily acts as an inhibitor of growth and metabolic processes.

Changes in ABA concentration internally or in the surroundingenvironment in contact with a plant results in modulation of many genesand gene products. Examples of such ABA responsive genes and geneproducts are shown in the Reference, Sequence, Protein Group, ProteinGroup Matrix tables, MA_diff, and MA_clust tables. These genes and/orproducts are responsible for effects on traits such as plant vigor andseed yield. They were discovered and characterized from a much largerset of genes by experiments designed to find genes whose mRNA productschanged in concentration in response to application of ABA to plants.

While ABA responsive polynucleotides and gene products can act alone,combinations of these polynucleotides also affect growth anddevelopment. Useful combinations include different ABA responsivepolynucleotides and/or gene products that have similar transcriptionprofiles or similar biological activities, and members of the same orsimilar biochemical pathways. Whole pathways or segments of pathways arecontrolled by transcription factor proteins and proteins controlling theactivity of signal transduction pathways. Therefore, manipulation ofsuch protein levels is especially useful for altering phenotypes andbiochemical activities of plants. In addition, the combination of an ABAresponsive polynucleotide and/or gene product with anotherenvironmentally responsive polynucleotide is also useful because of theinteractions that exist between hormone-regulated pathways, stress anddefence induced pathways, nutritional pathways and development. Here, inaddition to polynucleotides having similar transcription profiles and/orbiological activities, useful combinations include polynucleotides thatmay have different transcription profiles but which participate incommon or overlapping pathways.

Such ABA responsive genes and gene products can function to eitherincrease or dampen the above phenotypes or activities either in responseto changes in ABA concentration or in the absence of ABA fluctuations.The MA_diff Table(s) reports the transcript levels of the experiment(see EXPT ID: 108560, 108561, 108513, 108597). For transcripts that hadhigher levels in the samples than the control, a “+” is shown. A “−” isshown for when transcript levels were reduced in root tips as comparedto the control. For more experimental detail see the Example sectionbelow.

ABA genes are those sequences that showed differential expression ascompared to controls, namely those sequences identified in the MA_difftables with a “+” or “−” indication.

ABA Genes Identified by Cluster Analyses of Differential Expression

ABA Genes Identified by Correlation to Genes that are DifferentiallyExpressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of ABA genes is any group in the MA_clust thatcomprises a cDNA ID that also appears in Expt ID 108560, 108561, 108513,108597 of the MA_diff table(s).

ABA Genes Identified by Correlation to Genes that Cause PhysiologicalConsequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of ABAgenes. A group in the MA_clust is considered a ABA pathway or network ifthe group comprises a cDNA ID that also appears in Knock-in or Knock-outtables that causes one or more of the phenotypes described in sectionabove.

ABA Genes Identified by Amino Acid Sequence Similarity

ABA genes from other plant species typically encode polypeptides thatshare amino acid similarity to the sequences encoded by corn andArabidopsis ABA genes. Groups of ABA genes are identified in the ProteinGroup table. In this table, any protein group that comprises a peptideID that corresponds to a cDNA ID member of a ABA pathway or network is agroup of proteins that also exhibits ABA functions/utilities.

Further, promoters of ABA responsive genes, as described in theReference tables, for example, are useful to modulate transcription thatis induced by ABA or any of the following phenotypes or biologicalactivities below.

III.C.1.a. Use of Abscissic Acid Responsive Genes to Modulate Phenotypes

ABA responsive genes and gene products are useful to or modulate one ormore of the following phenotypes including development such as cellgrowth (promotion of leaf cell elongation), fruit development (fruitdrop and inhibition of parthenocarpy and ovary growth), seed development(maturation of zygotic and somatic embryos, embryo development, seeddevelopment and maturation, acquisition of desiccation tolerance,dormancy including control rate and timing of germination, prolongationof seed storage and viability, and inhibition of hydrolytic enzymesynthesis); growth of roots such as inhibition of root elongation underlow water potential), stems, buds (such as promotion of dormancy andlateral/axillary bud formation), leaves, and inhibition of aba-inducedgrowth and elongation; biomass (such as fresh and dry weight during anytime in plant life, such as maturation), number, size, and weight offlowers and seeds); senescence (including abscission, leaf fall, andflower longevity); differentiation (including plastid/chloroplastdifferentiation and regulation of sterility); and stress responses (suchas mediation of response to desiccation, drought, salt and cold).

To regulate any of the phenotype(s) above, activities of one or more ofthe ABA responsive genes or gene products can be modulated in anorganism and tested by screening for the desired trait. Specifically,the gene, mRNA levels, or protein levels can be altered in a plantutilizing the procedures described herein and the phenotypes can beassayed. As an example, a plant can be transformed according to Bechtoldand Pelletier (1998, Methods. Mol. Biol. 82:259-266) and/or screened forvariants as in Winkler et al. (1998) Plant Physiol 118: 743-50 andvisually inspected for the desired phenotype or metabolically and/orfunctionally assayed according to Koorneef and Karssen (1994, Seeddormancy and germination, In: Arabidopsis, Cold Spring Harbor Lab.Press, pp 314-334), Cramer et al (1998, J. Exptl. Botany 49:191-198),and White and Rivin (2000, Plant Physiol 122: 1089-97). Phillips et al.(1997) EMBO J 16: 4489-96; Nambara et al (1995) Development 121:629-636; Hays et al (1999) Plant Physiol. 119: 1065-72; Filonova et al(2000) J Exptl Botany 51: 249-64; White et al (2000) Plant Physiol. 122:1081-88; and Visser et al. (1998) Plant Mol Biol 37: 131-40; Rohde etal. (2000) Plant Cell 12:35-52; and Cramer et al. (1998) J. experimentalBotany. 49: 191-198.

III.C.1.b. Use of Abscissic Acid Responsive Genes to ModulateBiochemical Activities

The activities of one or more of the ABA responsive genes can bemodulated to change biochemical or metabolic activities and/or pathwayssuch as those noted below. Such biological activities can be measuredaccording to the citations included in the Table below:

BIOCHEMICAL OR METABOLIC ACTIVITIES PROCESS AND/OR PATHWAYS CITATIONSINCLUDING ASSAYS Growth, Farnesylation Pei Et Al (1998) Science 282:287-290; Differentiation And Cutler Et Al. (1996) Science Development273: 1239 Nitrogen Metabolism Goupil Et Al (1998) J Exptl Botany 49:1855-62 Water Conservation Stomatal Development Allen Et Al. (1999)Plant Cell 11: And Resistance To And Physiology 1785-1798 Drought AndOther Li Et Al. 2000 Science 287: 300-303 Related Stresses Burnett Et Al2000. J. Exptl Botany 51: 197-205 Raschke (1987) In: Stomatal FunctionZeiger Et Al. Eds., 253-279 Stress Response Pathways Bush And Pages(1998) Plant Mol. Biol. 37: 425-35 Inhibition Of Ethylene Spollen Et Al(2000) Plant Physiol. Production Under Low 122: 967-976 Water PotentialProline And Other Hare Et Al. (1998) Plant, Cell And Osmolite SynthesisAnd Environment 21: 535-553; Hare Et Al. Degradation (1999) J. Exptl.Botany 50: 413-434 Plasmalemma And Macrobbie (1998) Philos Trans R SocTonoplast Ion Channel Lond B Biol Sci 353: 1475-88; Li Et Changes Al(2000) Science 287: 300-303; Barkla Et Al. (1999) Plant Physiol. 120:811-819 Ca2+ Accumulation Lacombe Et Al. (2000) Plant Cell 12: 837-51;Wang Et Al. (1998) Plant Physiol 118: 1421-1429; Shi Et Al. (1999) PlantCell 11: 2393-2406 K+ Efflux Gaymard Et Al. (1998) Cell 94: 647-655Activation Of Kinases Jonak Et Al. (1996) Proc. Natl. Acad. AndPhosphatases Sci 93: 11274-79; Sheen (1998) Proc. Natl. Acad. Sci. 95:975-80; Allen Et Al. (1999) Plant Cell 11: 1785-98

Other biological activities that can be modulated by the ABA responsivegenes and gene products are listed in the Reference tables. Assays fordetecting such biological activities are described in the Protein Domaintable.

ABA responsive genes are characteristically differentially transcribedin response to fluctuating ABA levels or concentrations, whetherinternal or external to an organism or cell. The MA_diff reports thechanges in transcript levels of various ABA responsive genes in entireseedlings at 1 and 6 hours after a plant was sprayed with a Hoagland'ssolution enriched with ABA as compared to seedlings sprayed withHoagland's solution only.

The data from this time course can be used to identify a number of typesof ABA responsive genes and gene products, including “early responders,”and “delayed ABA responders”, “early responder repressors” and “delayedrepressors”. Profiles of these different ABA responsive genes are shownin the Table below together with examples of the kinds of associatedbiological activities.

EXAMPLES OF TRANSCRIPT TYPE OF PHYSIOLOGICAL BIOCHEMICAL LEVELS GENESCONSEQUENCES ACTIVITY Up Regulated Early Responders ABA PerceptionTranscription Factors Transcripts To ABA ABA Uptake Transporters (LevelAt 1 Hr ≅6 Hr) Modulation Of ABA Change In Cell Membrane or ResponseStructure (Level At 1 Hr > 6 Hr) Transduction Kinases And PhosphatasesPathways Transcription Activators Specific Gene Change In ChromatinTranscription Structure And/Or Initiation Localized DNA Topology UpRegulated Delayed Maintenance Of Transcription Factors TranscriptsResponders Response To ABA Specific Factors (Initiation (Level At 1 Hr <6 Hr) Maintenance Of Seed And Elongation) For Dormancy, Stress ProteinSynthesis Stomatal Closure, Maintenance Of Mrna Water Uptake StabilityControl, Abscission Maintenance Of Protein And Senescence StabilityControl Pathways Maintenance Of Protein- Protein InteractionDown-Regulated Early Responder Negative Regulation Transcription FactorsTranscripts Repressors Of Of ABA Pathways Change In Protein (Level At 1Hr ≅ 6 Hr) ABA State Of Released Structure By or Metabolism Changes InPathways Phosphorylation (Kinases) (Level At 6 Hr > 1 Hr) Genes With AndProcesses Or Dephosphoryaltion Discontinued Operating In Cells(Phosphatases) Expression Or Change In Chromatin UnsTable mRNA StructureAnd/Or DNA In Presence Of Topology ABA Down-Regulated Delayed NegativeRegulation Transcription Factors Transcripts Repressors Of Of ABAPathways Kinases And Phosphatases (Level At 1 Hr > 6 Hr) ABA State OfReleased Stability Of Factors For Metabolism Maintenance Of ProteinSynthesis And Genes With Pathways Released Degradation Discontinued FromRepression Expression Or UnsTable mRNA Changes In Pathways In PresenceOf And Processes ABA Operating In Cells

Use of Promoters of ABA Responsive Genes

Promoters of ABA responsive genes are useful for transcription of anydesired polynucleotide or plant or non-plant origin. Further, anydesired sequence can be transcribed in a similar temporal, tissue, orenvironmentally specific patterns as the ABA responsive genes where thedesired sequence is operably linked to a promoter of a ABA responsivegene. The protein product of such a polynucleotide is usuallysynthesized in the same cells, in response to the same stimuli as theprotein product of the gene from which the promoter was derived. Suchpromoter are also useful to produce antisense mRNAs to down-regulate theproduct of proteins, or to produce sense mRNAs to down-regulate mRNAsvia sense suppression.

III.C.2. Auxin Responsive Genes, Gene Components and Products

Plant hormones are naturally occurring substances, effective in verysmall amounts that stimulate or inhibit growth or regulate developmentalprocesses in plants. One of the plant hormones is indole-3-acetic acid(IAA), often referred to as Auxin.

Changes in Auxin concentration in the surrounding environment in contactwith a plant or in a plant results in modulation of the activities ofmany genes and hence levels of gene products. Examples of such Auxinresponsive genes and their products are shown in the Reference andSequence Tables. These genes and/or products are responsible for effectson traits such as plant vigor and seed yield. The genes were discoveredand characterized from a much larger set by experiments designed to findgenes whose mRNA products changed in response to application of Auxin toplants.

Manipulation of one or more Auxin responsive gene activities are usefulto modulate the biological activities and/or phenotypes listed below.Auxin response genes and gene products can act alone or in combination.Useful combinations include Auxin response genes and/or gene productswith similar transcription profiles, similar biological activities, ormembers of the same or functionally related biochemical pathways. Wholepathways or segments of pathways are controlled by transcription factorproteins and proteins controlling the activity of signal transductionpathways. Therefore, manipulation of the levels of such proteins isespecially useful for altering phenotypes and biochemical activities ofplants. The MA_diff Table(s) reports the transcript levels of theexperiment (see EXPT ID: 108564, 108565, 108516, 108554, 108466, 107886,107891, SMD 3743, and NAA (relating to SMD 3749, SMD 6338, SMD 6339)).For transcripts that had higher levels in the samples than the control,a “+” is shown. A “−” is shown for when transcript levels were reducedin root tips as compared to the control. For more experimental detailsee the Example section below.

NAA genes are those sequences that showed differential expression ascompared to controls, namely those sequences identified in the MA_difftables with a “+” or “−” indication.

NAA Genes Identified by Cluster Analyses of Differential Expression

NAA Genes Identified by Correlation to Genes that are DifferentiallyExpressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of NAA genes is any group in the MA_clust thatcomprises a cDNA ID that also appears in Expt ID 108564, 108565, 108516,108554, 108466, 107886, 107891, SMD 3743, and NAA (relating to SMD 3749,SMD 6338, SMD 6339) of the MA_diff table(s).

NAA Genes Identified by Correlation to Genes that Cause PhysiologicalConsequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of NAAgenes. A group in the MA_clust is considered a NAA pathway or network ifthe group comprises a cDNA ID that also appears in Knock-in or Knock-outtables that causes one or more of the phenotypes described in sectionabove.

NAA Genes Identified by Amino Acid Sequence Similarity

NAA genes from other plant species typically encode polypeptides thatshare amino acid similarity to the sequences encoded by corn andArabidopsis NAA genes. Groups of NAA genes are identified in the ProteinGroup table. In this table, any protein group that comprises a peptideID that corresponds to a cDNA ID member of a NAA pathway or network is agroup of proteins that also exhibits NAA functions/utilities.

Such Auxin responsive genes and gene products can function to eitherincrease or dampen the above phenotypes or activities either in responseto changes in Auxin concentration or in the absence of Auxinfluctuations. Further, promoters of Auxin responsive genes, as describedin the Reference tables, for example, are useful to modulatetranscription that is induced by Auxin or any of the followingphenotypes or biological activities below.

III.C.2.a. Use of Auxin Responsive Genes, Gene Components and Productsto Modulate Phenotypes

Auxin responsive genes and gene products are useful to or modulate oneor more phenotypes including growth, apical dominance, vascular growth,roots, inhibition of primary root elongation, increased lateral rootformation, stems, lateral buds, lateral branching, reduction ofbranching, for high density growth per acre, for increased woodproduction, lateral organ initiation and/or positioning in apicalmeristem, organ formation, for example, fruit number in tomatoes,leaves, height/stature, e.g., taller crops or increase wood production,regeneration and differentiation of cultured cells or plantlets,biomass, fresh and dry weight during any time in plant life, such asmaturation; number of flowers; number of seeds; number of branches;number of leaves; starch content, seed yield, including number, size,weight, harvest index, starch content, fruit yield, number, size,weight, harvest index, starch content, development, orienting cellgrowth, establishment and maintenance of plant axis, apical dominance,cell plate placement, polarised growth, initiation and/or development,of embryos morphogenic progression, e.g., from early radial to lateaxialized torpedo stages, differentiation of cells into morphologicallydifferent cell layers, cotyledon separation, fruit development,abscission, leading to modulation of fruit drop, parthenocarpy, seedlesscrops resulting from lack of seed set, vascularization, e.g. hypocotyland cotyledon tissues, genetic control of vascular patterning andinfluences its maturation; specification of the sites where vasculardifferentiation will occur; determination of the direction and extent ofvascular tissue formation, maintenance of the continuity of vasculardevelopment with plant growth, tropic responses, gravitropic responses,e.g. affecting roots and shoots, and modulation of phototropicsensitivity, e.g. increase growth under a reduced light spectrum.

Further, any desired sequence can be transcribed in similar temporal,tissue, or environmentally specific patterns as the Auxin responsivegenes when the desired sequence is operably linked to a promoter of anAuxin responsive gene.

To modulate any of the phenotype(s) above, activities of one or more ofthe Auxin response genes or gene products can be modulated and theplants can be tested by screening for the desired trait. Specifically,the gene, mRNA levels, or protein levels can be altered in a plantutilizing the procedures described herein and the phenotypes can bescreened for variants as in Winkler et al. (1998) Plant Physiol 118:743-50 and assayed, for example, in accordance with Bechtold andPelletier (1998). Methods Mol. Biol. 82: 259-266; Clough and Bent(1998). 16: 735-743; Krysan et al. (1999). Plant Cell 11:2283-2290.

III.C.2.b. Use of Auxin Responsive Genes, Gene Components and Productsto Biochemical Activities:

The activities of one or more of the Auxin responsive genes can bemodulated to change biochemical or metabolic activities and/or pathwayssuch as those noted below. Such biological activities are documented andcan be measured according to the citations included in the Table below:

BIOCHEMICAL OR METABOLIC ACTIVITIES CITATIONS INCLUDING PROCESS AND/ORPATHWAYS ASSAYS Cell Growth and Protein Ubiquitination Gray et al.(1999) Genes and Differentiation Develop, 13: 1678-1691 Bechtold andPelletier (1998). Methods. Mol. Biol. 82: 259-266 Cell Wall looseningand Catala et al. (2000). Plant Physiol. Expansion 122: 527-534.Cosgrove, D. (1993). New Phytol. 124: 1-23. Auxin/Cytokinin RatioChanging Auxin and/or Chen et al. (1988). Plant Physiol. cytokininsynthesis and/or 86: 822-825 turnover Tam et al. (2000). Plant Physiol.123: 589-595 Bartel and Fink. (1995). Science 268: 1745-1748. Prinsen etal. (1995). Quantifying phytohormones in transformed plants. In: Methodsin Molecular Biology. 44: 245-262. Auxin Transport Channeling of polarAuxin Reed et al. (1998). Plant Physiol. Transport 118: 1369-1378.Estelle, M. (1998). Plant Cell 10: 1775-1778 Auxin Efflux Between CellsReed et al. (1998). Plant Physiol. 118: 1369-1378. Marchant et al.(1999). EMBO J. 18: 2066-2073. Auxin Influx In and Out of a Reed et al.(1998). Plant Physiol. Cell 118: 1369-1378. Marchant et al. (1999). EMBOJ. 18: 2066-2073. Electogenic Proton Symport Young et al. (1999).Biochim of Auxin Biophys Acta. 1415(2): 306-22 Signal Transduction K+Accumulation Philippar et al. (1999). Proc. Natl. Acad. Sci. 96:12186-12191 Permeability of Cell Marchant et al. (1999). EMBO J.Membranes 18: 2066-2073. Guanine-Nucleotide Steinmann et al. (1999).Science Exchange 286: 316-318. Peyroche et al. (1996). Nature 384:479-481. Protein Phosphorylation Christensen et al. (2000). Cell 100:469-478. Hirt (2000). Proc. Natl. Acad Sci. 97: 2405-2407. Interactionwith Ethylene Madlung et al. (1999). Plant mode of action Physiol. 120:897-906. Xu et al. (1998). Plant Physiol. 118: 867-874. Protein TurnoverLocalization of Polypeptides Grebe et al. (2000). Plant Cell. with thebasal End of Cells 12: 343-356

Other biological activities that can be modulated to by the Auxinresponsive genes and their products are listed in the Reference Tables.Assays for detecting such biological activities are described in theDomain section of the Reference Tables.

Auxin responsive genes are characteristically differentially transcribedin response to fluctuating Auxin levels or concentrations, whetherinternal or external to an organism or cell. The MA_diff(s) report(s)the changes in transcript levels of various Auxin responsive genes inthe aerial parts of a seedling at 1 and 6 hours after the seedling wassprayed with a solution enriched with Auxin as compared to aerial partsof a seedling sprayed with water.

The data from this time course can be used to identify a number of typesof Auxin responsive genes and gene products, including “earlyresponders,” and “delayed responders.” Profiles of these differentclasses of Auxin responsive genes are shown in the Table below togetherwith examples of the kinds of associated biological activities.

EXAMPLES OF BIOCHEMICAL TRANSCRIPT TYPE OF PHYSIOLOGICAL ACTIVITY OFGENE LEVEL GENES CONSEQUENCES PRODUCTS Upregulated Early Auxinperception Transcription factors transcripts responders to AuxinTransporters; channeling (level at 1 hr Auxin Uptake/transport of polarAuxin transport ≅6 hours) Modulation of Kinases and (level at 1 hr > 6Auxin response phosphatases; protein hours) transduction ubiqutination;guanine pathways nucelotide exchange; Initiating changing Auxin and/ortranscription of cytokininin synthesis specific gene(s) and/or turnover;Modification of cell interaction with ethylene walls mode of actionModification of cell Auxin metabolic structures pathways Modification ofChange in chromatin metabolism structure and/or DNA topologyTranscriptional activators Change in activity of protein-proteininteractions Cell wall and cell growth promoting pathways Change inactivity of cytoskeletal proteins modulating cell structure Metabolicenzymes Coordination and control of central carbon and Auxin metabolismUpregulated “Delayed” Completion and/or Transcription factorstranscripts (level Responders Maintenance of Changes in membrane at 1 hr< 6 hr) Auxin response protein, membrane channel Initiating and/ortransporter protein transcription of activity specific gene(s) Change inchromatin Modification of cell structure and/or DNA walls topologyModification of cell Transcriptional activators structures Change inactivity of Modification of protein-protein interactions metabolism Cellwall proteins Change(s) in activity of cytoskeletal proteins modulatingcell structure Coordination and control of central carbon and Auxinmetabolism metabolic enzymes Downregulated Early repressor Repression ofTranscription factors transcripts responders to Auxin induced Changes inactivity of (level at 1 hour ≅ Auxin proteins released cytoskeletalproteins 6 hours) Genes for Reorientation of modulating cell structure(level at 1 hour > pathways metabolism in Changes in chromatin 6 hours)diminished in certain cells structure and/or DNA presence of topologyAuxin Changes in protein structure and/or function by phosphorylation(kinases) and/or dephosphorylation (phosphatases) Stability of factorsfor protein translation Changes in cell membrane structure Changes inchromatin and/or localized DNA topology Changes in protein- proteininteraction Metabolic enzymes Down-regulated “Delayed” Maintenance ofTranscription factors transcripts repressor Auxin stimulated Change inactivity of (level at 1 hour < responders to state(s) in certaincytoskeletal proteins 6 hours) Auxin cells modulating cell structureGenes for Reorientation of Changes in chromatin pathways metabolism instructure and/or DNA diminished in certain cells topology presence ofChanges in protein Auxin structure and/or function by phosphorylation(kinases) and/or dephosphorylation (phosphatases) Stability of factorsfor protein translation Changes in cell membrane structure Changes inchromatin and/or localized DNA topology Changes in protein- proteininteraction Metabolic enzymes

Use of Promoters of NAA Responsive Genes

Promoters of NAA responsive genes are useful for transcription of anydesired polynucleotide or plant or non-plant origin. Further, anydesired sequence can be transcribed in a similar temporal, tissue, orenvironmentally specific patterns as the NAA responsive genes where thedesired sequence is operably linked to a promoter of a NAA responsivegene. The protein product of such a polynucleotide is usuallysynthesized in the same cells, in response to the same stimuli as theprotein product of the gene from which the promoter was derived. Suchpromoter are also useful to produce antisense mRNAs to down-regulate theproduct of proteins, or to produce sense mRNAs to down-regulate mRNAsvia sense suppression.

III.C.3. Brassinosteroid Responsive Genes, Gene Components and Products:

Plant hormones are naturally occurring substances, effective in verysmall amounts, which act as signals to stimulate or inhibit growth orregulate developmental processes in plants. Brassinosteroids (BRs) arethe most recently discovered, and least studied, class of planthormones. The major physiological response affected by BRs is thelongitudinal growth of young tissue via cell elongation and possiblycell division. Consequently, disruptions in BR metabolism, perceptionand activity frequently result in a dwarf phenotype. In addition,because BRs are derived from the sterol metabolic pathway, anyperturbations to the sterol pathway can affect the BR pathway. In thesame way, perturbations in the BR pathway can have effects on the laterpart of the sterol pathway and thus the sterol composition of membranes.

Changes in BR concentration in the surrounding environment or in contactwith a plant result in modulation of many genes and gene products.Examples of such BR responsive genes and gene products are shown in theReference and Sequence Tables. These genes and/or products areresponsible for effects on traits such as plant biomass and seed yield.These genes were discovered and characterized from a much larger set ofgenes by experiments designed to find genes whose mRNA abundance changedin response to application of BRs to plants.

While BR responsive polynucleotides and gene products can act alone,combinations of these polynucleotides also affect growth anddevelopment. Useful combinations include different BR responsivepolynucleotides and/or gene products that have similar transcriptionprofiles or similar biological activities, and members of the same orfunctionally related biochemical pathways. Whole pathways or segments ofpathways are controlled by transcription factors and proteinscontrolling the activity of signal transduction pathways. Therefore,manipulation of such protein levels is especially useful for alteringphenotypes and biochemical activities of plants. In addition, thecombination of a BR responsive polynucleotide and/or gene product withanother environmentally responsive polynucleotide is useful because ofthe interactions that exist between hormone-regulated pathways, stresspathways, nutritional pathways and development. Here, in addition topolynucleotides having similar transcription profiles and/or biologicalactivities, useful combinations include polynucleotides that may havedifferent transcription profiles but which participate in common oroverlapping pathways. The MA_diff Table(s) reports the transcript levelsof the experiment (see EXPT ID: 108580, 108581, 108557, 108478, 108479,108480, 108481). For transcripts that had higher levels in the samplesthan the control, a “+” is shown. A “−” is shown for when transcriptlevels were reduced in root tips as compared to the control. For moreexperimental detail see the Example section below.

BR genes are those sequences that showed differential expression ascompared to controls, namely those sequences identified in the MA_difftables with a “+” or “−” indication.

BR Genes Identified by Cluster Analyses of Differential Expression

BR Genes Identified by Correlation to Genes that are DifferentiallyExpressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of BR genes is any group in the MA_clust thatcomprises a cDNA ID that also appears in Expt ID 108580, 108581, 108557,108478, 108479, 108480, 108481 of the MA_diff table(s).

BR Genes Identified by Correlation to Genes that Cause PhysiologicalConsequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of BR genes.A group in the MA_clust is considered a BR pathway or network if thegroup comprises a cDNA ID that also appears in Knock-in or Knock-outtables that causes one or more of the phenotypes described in sectionabove.

BR Genes Identified by Amino Acid Sequence Similarity

BR genes from other plant species typically encode polypeptides thatshare amino acid similarity to the sequences encoded by corn andArabidopsis BR genes. Groups of BR genes are identified in the ProteinGroup table. In this table, any protein group that comprises a peptideID that corresponds to a cDNA ID member of a BR pathway or network is agroup of proteins that also exhibits BR functions/utilities.

Such BR responsive genes and gene products can function to eitherincrease or dampen the above phenotypes or activities either in responseto changes in BR concentration or in the absence of BR fluctuations.Further, promoters of BR responsive genes, as described in the Referencetables, for example, are useful to modulate transcription that isinduced by BR or any of the following phenotypes or biologicalactivities below.

III.C.3.a. Use of Brassinosteroid Responsive Genes to ModulatePhenotypes

Brassinosteroid responsive genes and gene products are useful tomodulate one or more phenotypes including growth (promotes cellelongation, elongation accelerated at low temperatures for increasedplant growth in marginal lands, acts in concert with other hormones topromote cell division); roots (inhibitory to root growth, and expressionin roots would inhibit bud breaking due to higher auxin:cytokinin ratioin epicotyl); stems (inhibits radial growth while causing stemelongation, in low concentrations, promotes radial expansion, andincreases biomass); height; seeds; promotes cell expansion in embryo andthus enhances germination; leaves; increase biomass; flowers, increasereproduction; biomass; fresh and dry weight during any time in plantlife, such as maturation; number of flowers; number of seeds; number ofbranches; number of leaves; starch content; seed yield (includingnumber, size, weight, harvest index, starch content; fruit yield,number, size, weight, harvest index, and starch content); development;morphogenesis; control of organ size and shape; development of newornamentals; control of leaf size and shape; promotes leaf unrolling andenlargement; for development of new leafy ornamentals; seed development;inhibition of de-etiolation; dormancy; accelerated germination at lowtemperatures; root; gravitropism; senescence; promoted in light grownplants; inhibiting synthesis or perception could extend life span ofdesired tissues/organs; differentiation; vascularization; promotes xylemdifferentiation; increases xylem fiber length; resistance responses;increases resistance to pathogens; and tropic responses.

Gravitropic Responses Affecting Roots

Further, any desired sequence can be transcribed in similar temporal,tissue, or environmentally specific patterns as the BR responsive geneswhen the desired sequence is operably linked to a promoter of a BRresponsive gene.

To improve any of the desired phenotype(s) above, activities of one ormore of the BR response genes or gene products can be modulated and theplants tested by screening for the desired trait. Specifically, thegene, mRNA levels, or protein levels can be altered in a plant utilizingthe procedures described herein and the phenotypes can be assayed. As anexample, a plant can be transformed according to Bechtold and Pelletier(1998, Methods. Mol. Biol. 82:259-266, and/or screened for variants asin Winkler et al. (1998) Plant Physiol 118: 743-50, visually inspectedfor the desired phenotype and metabolically and/or functionally assayedaccording to Choe et al. (1999, Plant Cell 11:207-21 and Plant Physiol119: 897-907), Yamamoto et al. (1997, Plant Cell Physiol 38:980-3),Asami and Yshida (1999, Trends in Plant Sciences, 4:348-353) and Azpirozet al. (1998, Plant Cell 10:219-230)

III.C.3.b. Use of Brassinosteroid Responsive Genes to ModulateBiochemical Activities

The activities of one or more of the BR responsive genes can bemodulated to change biochemical or metabolic activities and/or pathwayssuch as those noted below. Such biological activities are documented andcan be measured according to the citations included in the Table below:

BIOCHEMICAL OR CITATIONS METABOLIC ACTIVITIES INCLUDING PROCESS AND/ORPATHWAYS ASSAYS BR Transport BR Efflux Between Cells B. Schulz and K.Feldmann, unpub. results BR Influx In And Out Of A B. Schulz and K. CellFeldmann, unpub. results Signal Permeability Of Cell TransductionMembranes Protein Phosphorylation Metabolism Major Growth CoordinatingPathways

Other biological activities that can be modulated by the BR responsivegenes and gene products are listed in the Reference Tables. Assays fordetecting such biological activities are described in the Domain sectionof the Reference Tables.

BR responsive genes are differentially transcribed in response tofluctuating BR levels or concentrations, whether internal or external toan organism or cell. The MA_diff table(s) report(s) the changes intranscript levels of various BR responsive genes in the aerial parts ofa seedling at 1 and 6 hours after a plant was sprayed with a solutionenriched with BR as compared to seedlings sprayed with water. The datafrom this time course can be used to identify a number of types of BRresponsive genes and gene products, including “early responders,”“delayed responders.” Profiles of these different categories of BRresponsive genes are shown in the Table below together with examples ofthe kinds of associated biological activities.

EXAMPLES OF TRANSCRIPT PHYSIOLOGICAL BIOCHEMICAL LEVELS TYPE OF GENESCONSEQUENCES ACTIVITY Up Regulated Early BR Perception TranscriptionTranscripts Responders To Factors (Level At 1 Hr ≈ 6 Hr) BR BR TransportReceptors (Level At 1 Hr > 6 Hr) Transporters Change In Cell MembraneStructure BR Biosynthesis Feedback Regulated Feedback Biosynthetic GenesKinases And Phosphatases Modulation Of 2^(nd) Messengers, Eg., BRResponse Calmodulin Transduction Pathways Specific Gene TranscriptionTranscription Activators Initiation Change In Chromatin Structure And/OrLocalized DNA Topology Up Regulated Delayed Maintenance Of TranscriptionTranscripts Responders Response To Br Factors (Level At 1 Hr < 6 Hr) BRBiosynthetic Genes Specific Factors (Initiation And Elongation) ForProtein Synthesis Maintenance Of Mrna Stability Maintenance Of ProteinStability Maintenance Of Protein-Protein Interaction Cell And Organ CellWall Elongation Elongation Gravitropism Down-Regulated Early ResponderNegative Transcription Transcripts Repressors Of BR Regulation OfFactors (Level At 1 Hr ≈ 6 Hr) State Of Metabolism BR Pathways Change InProtein (Level At 6 Hr > 1 Hr) Genes With Released Structure ByDiscontinued Changes In Phosphorylation Expression Or Pathways And(Kinases) Or UnsTable Mrna In Processes Dephosphoryaltion Presence OfOperating In (Phosphatases) BR Cells Change In Chromatin StructureAnd/Or DNA Topology Down-Regulated Delayed Negative TranscriptionTranscripts Repressors Of Regulation Of Factors (Level At 1 Hr > 6 Hr)BR State Of BR Pathways Kinases And Metabolism Released PhosphatasesGenes With Maintenance Of Stability Of Factors Discontinued Pathways ForProtein Expression Or Released From Synthesis And UnsTable MrnaRepression Degradation In Presence Of Changes In BR Pathways AndProcesses Operating In Cells

Use of Promoters of BR Responsive Genes

Promoters of BR responsive genes are useful for transcription of anydesired polynucleotide or plant or non-plant origin. Further, anydesired sequence can be transcribed in a similar temporal, tissue, orenvironmentally specific patterns as the BR responsive genes where thedesired sequence is operably linked to a promoter of a BR responsivegene. The protein product of such a polynucleotide is usuallysynthesized in the same cells, in response to the same stimuli as theprotein product of the gene from which the promoter was derived. Suchpromoter are also useful to produce antisense mRNAs to down-regulate theproduct of proteins, or to produce sense mRNAs to down-regulate mRNAsvia sense suppression.

III.C.4. Cytokinin Responsive Genes, Gene Components and Products

Plant hormones are naturally occurring substances, effective in verysmall amounts, which act as signals to stimulate or inhibit growth orregulate developmental processes in plants. Cytokinins (BA) are a groupof hormones that are best known for their stimulatory effect on celldivision, although they also participate in many other processes andpathways. All naturally occurring BAs are aminopurine derivatives, whilenearly all synthetic compounds with BA activity are 6-substitutedaminopurine derivatives. One of the most common synthetic BAs used inagriculture is benzylaminopurine (BAP).

Changes in BA concentration in the surrounding environment or in contactwith a plant results in modulation of many genes and gene products.Examples of such BA responsive genes and gene products are shown in theReference, Sequence, Protein Group, Protein Group Matrix tables, MA_diffand MA_clust. These genes and/or products are responsible for effects ontraits such as plant vigor and seed yield. They were discovered andcharacterized from a much larger set by experiments designed to findgenes whose mRNA products changed in response to application of BA toplants.

While cytokinin responsive polynucleotides and gene products can actalone, combinations of these polynucleotides also affect growth anddevelopment. Useful combinations include different BA responsivepolynucleotides and/or gene products that have similar transcriptionprofiles or similar biological activities, and members of the same orfunctionally related biochemical pathways. Whole pathways or segments ofpathways are controlled by transcription factor proteins and proteinscontrolling the activity of signal transduction pathways. Therefore,manipulation of such protein levels is especially useful for alteringphenotypes and biochemical activities of plants. In addition, thecombination of a BA responsive polynucleotide and/or gene product withanother environmentally responsive polynucleotide is also useful becauseof the interactions that exist between hormone-regulated pathways,stress pathways, nutritional pathways and development. Here, in additionto polynucleotides having similar transcription profiles and/orbiological activities, useful combinations include polynucleotides thatmay have different transcription profiles but which participate incommon or overlapping pathways. The MA_diff Table(s) reports thetranscript levels of the experiment (see EXPT ID: 108566, 108567,108517). For transcripts that had higher levels in the samples than thecontrol, a “+” is shown. A “−” is shown for when transcript levels werereduced in root tips as compared to the control. For more experimentaldetail see the Example section below.

BA genes are those sequences that showed differential expression ascompared to controls, namely those sequences identified in the MA_difftables with a “+” or “−” indication.

BA Genes Identified by Cluster Analyses of Differential Expression

BA Genes Identified by Correlation to Genes that are DifferentiallyExpressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of BA genes is any group in the MA_clust thatcomprises a cDNA ID that also appears in Expt ID 108566, 108567, 108517of the MA_diff table(s).

BA Genes Identified by Correlation to Genes that Cause PhysiologicalConsequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of BA genes.A group in the MA_clust is considered a BA pathway or network if thegroup comprises a cDNA ID that also appears in Knock-in or Knock-outtables that causes one or more of the phenotypes described in sectionabove.

BA Genes Identified by Amino Acid Sequence Similarity

BA genes from other plant species typically encode polypeptides thatshare amino acid similarity to the sequences encoded by corn andArabidopsis BA genes. Groups of BA genes are identified in the ProteinGroup table. In this table, any protein group that comprises a peptideID that corresponds to a cDNA ID member of a BA pathway or network is agroup of proteins that also exhibits BA functions/utilities.

Such BA responsive genes and gene products can function to eitherincrease or dampen the above phenotypes or activities either in responseto changes in BA concentration or in the absence of BA fluctuations.

Further, promoters of BA responsive genes, as described in the Referencetables, for example, are useful to modulate transcription that isinduced by BA or any of the following phenotypes or biologicalactivities below.

III.C.4.a. Use of Ba-Responsive Genes to Modulate Phenotypes

BA responsive genes and gene products are useful to or modulate one ormore phenotypes including growth, roots (such as inhibition ofelongation of root); stems (such as inhibition of elongation ofhypocotyl); lateral buds (such as promotion of outgrowth for rapidproduction of multiple shoots as a source for grafting); leaves such asdevelopment (including cell growth, such as expansion of cotyledon andpromotes cell enlargement for increased yield from leaf crops,chloroplast development such as delayed degradation of chloroplasts forincreased photosynthesis and crop yield, cell division and senescencesuch as delays for delayed conversion from photosynthesis to salvageprograms in leaves and for increased crop yield); differentiation suchas regulation of morphogenesis for manipulating callus growth andshoot/root formation in culture; maintenance of shoot meristem such asfor increased usable wood production, and reduced tiller number fordenser crop planting regimes; nutrient metabolism for effects on seedsize and effects on rate of seed set for increased seed yield; inductionof ethylene biosynthesis for control of fruit ripening; andparthenocarpy for control of sexual reproduction and production ofseedless fruits.

To regulate any of the phenotype(s) above, activities of one or more ofthe BA responsive genes or gene products can be modulated and the plantstested by screening for the desired trait. Specifically, the gene, mRNAlevels, or protein levels can be altered in a plant utilizing theprocedures described herein and the phenotypes can be assayed. As anexample, a plant can be transformed according to Bechtold and Pelletier(1998, Methods. Mol. Biol. 82:259-266) and/or screened for variants asin Winkler et al. (1998) Plant Physiol 118: 743-50 and visuallyinspected for the desired phenotype or molecularly or metabolically orfunctionally assayed according to Lohman et al (1994, Physil. Plant92:322-328), Woolhouse (1983, In Agricultural Research-Strategies ofPlant reproduction, Meudt, ed., 201-236), Medford et al. (1989, PlantCell 1: 403-13), Vogel et al. (1998, Genetics 149:417-27), Ehnes andRoitsch (1997, Plant J 1: 539-48), Rotino et al. (1997, Nat. Biotchnol.15: 1398-1401).

III.C.4.b. Use of Ba-Responsive Genes to Modulate Biochemical Activities

The activities of one or more of the BA responsive genes can bemodulated to change biochemical or metabolic activities and/or pathwayssuch as those noted below. Such biological activities can be measuredaccording to the citations included in the Table below:

BIOCHEMICAL OR METABOLIC ACTIVITIES AND/ CITATIONS PROCESS OR PATHWAYSINCLUDING ASSAYS Chloroplast Photosynthesis Benkova et al (1999) PlantFunctioning Physil 121: 245-252 Induction And Cell Cycle PhaseRiou-Khamlichi et al. Maintenance Transition (1999) Science 283: 1541-44Of Cell Division Senescence Cell Death/Apoptosis Lohman et al. (1994)Physiol Plant 92: 322-328 Signal Sensing Endogenous Kakimoto (1996)Science Transduction Stimuli To Trigger 274: 982-985 Growth And ShootFormation

Other biological activities that can be modulated by the BA responsivegenes and gene products are listed in the Reference tables. Assays fordetecting such biological activities are described in the Domain sectionabove.

BA responsive genes are characteristically differentially transcribed inresponse to fluctuating BA levels or concentrations, whether internal orexternal to an organism or cell. The MA_diff table reports the changesin transcript levels of various BA responsive genes in the aerial partsof a seedling at 1 and 6 hours after a plant was sprayed with aHoagland's solution enriched with BA as compared to seedlings sprayedwith Hoagland's solution only.

The data from this time course can be used to identify a number of typesof BA responsive genes and gene products, including “early responders,”and “delayed responders.” Profiles of these different BA responsivegenes are shown in the Table below together with examples of the kindsof associated biological activities.

GENE FUNCTIONAL TYPE OF EXAMPLES OF EXPRESSION CATEGORY OF BIOLOGICALBIOCHEMICAL LEVELS GENES ACTIVITY ACTIVITY Up Regulated Early BAPerception Transcription Factors Transcripts Responders To BA UptakeTransporters (Level At 1 h ≅ 6 h) BA Modulation Of BA Kinase,Receptor-Like Or Response Protein Kinase (Higher At 1 h TransductionThan 6 h) Pathways Specific Gene Ovule-Specific Homeotic TranscriptionProtein, Secretory Initiation Pathway Initiate And Cell Division ControlCoordinate Cell Protein, Cyclins, Cyclin- Division Dependent ProteinKinase (Cdpk), Cell Cycle Phosphatases, Mitosis-Specific ChromosomeSegregation Protein, Mitotic Phosphoprotein, Dna Replication Proteins,Helicase Telomerase, Centromere Protein, tRNA Synthase Regulation OfSenescence-Associated Pathways To Protein, Bifunctional SenescenceNuclease, Aba Pathway Genes, Ethylene Pathway Genes, Proteases,Nucleases, Pcd Genes Modulation Of Calvin Cycle, Chloroplast GeneChlorophyll A/B Binding Expression And Protein (Cab), PhotosysthesisTransketolase, Lipoxygenase, Chloroplast Rna Processing Protein,Chloroplast Envelope Membrane Protein. Modulation Of Glutamate Synthase,Photorespiration And Gogat, Asparagine Primary Nitrogen Synthase,Catalase, Assimilation In Peroxidase Leaves Expression Stress ResponseHeat Shock Proteins, Gst Wax Biosynthesis Fatty Acid Elongase- LikeProtein, Very-Long- Chain Fatty Acid Condensing Enzyme, Coa SynthaseNutrient Metabolism Vicilin Storage Protein Embryogenesis HomeoboxDomain Proteins Glycolysis, Mutase, Gluconeogenesis PhosphoglycerateMutase Ripening Pectate Lyase, Ethylene Pathway Genes Upregulated BALate BA Responsive Transfactors, Kinases, Transcripts RespondersPathways Phosphatases, LRR's, (Higher At 6 h Dna Remodelling Than 1 h)Proteins, Cu-Binding Proteins Cell Wall Extension Expansins, Extensins,Proline Rich Proteins Organogenesis AP2 Domain Containing ProteinsModulate Activation Transfactors Interacting Of Disease Defense WithResistant Genes Genes Modulate Responses Glycin-Rich Proteins, ToExternal Stimuli Wall-Associated Receptor Kinase (Wak) Osmotic StressProline Oxidase Tolerance Down-Regulated Repressors Of BA Regulation OfTransfactors (Such As Transcripts (Low Pathway Senescence-RelatedZinc-Finger Type), At Both 1 h and Gene Expression Kinases,Phosphatases, 6 h) G-Proteins, LRR Proteins, DNA Remodeling ProteinCarbonyl Reductases Regulation Of Genes Atpases Involved In OxygenaseMaintenance Of Octaprenyltransferase Apical Dominance. Auxin PathwayGenes Auxin Binding Proteins

Further, any desired sequence can be transcribed in similar temporal,tissue, or environmentally specific patterns as the BA responsive geneswhen the desired sequence is operably linked to a promoter of a BAresponsive gene.

III.C.5. Gibberellic Acid Responsive Genes, Gene Components and Products

Plant hormones are naturally occurring substances, effective in verysmall amounts, which act as signals to stimulate or inhibit growth orregulate developmental processes in plants. Gibberellic acid (GA) is ahormone in vascular plants that is synthesized in proplastids (givingrise to chloroplasts or leucoplasts) and vascular tissues. The majorphysiological responses affected by GA are seed germination, stemelongation, flower induction, anther development and seed and pericarpgrowth. GA is similar to Auxins, cytokinins and gibberellins, in thatthey are principally growth promoters.

Changes in GA concentration in the surrounding environment or in contactwith a plant result in modulation of many genes and gene products.Examples of such GA responsive genes and gene products are shown in theReference and Sequence Tables. These genes and/or products areresponsible for effects on traits such as plant vigor and biomass andseed yield. They were discovered and characterized from a much largerset of genes by experiments designed to find genes whose mRNA productschanged in concentration in response to application of nitrogen toplants.

While GA responsive polynucleotides and gene products can act alone,combinations of these polynucleotides also affect growth anddevelopment. Useful combinations include different GA responsivepolynucleotides and/or gene products that have similar transcriptionprofiles or similar biological activities, and members of the same orsimilar biochemical pathways. Whole pathways and/or segments of pathwaysare controlled by transcription factors and proteins that affect theactivity of signal transduction pathways. Therefore, manipulation ofsuch protein levels is especially useful for altering phenotypes andbiochemical activities of plants. In addition, the combination of a GAresponsive polynucleotide and/or gene product with anotherenvironmentally responsive polynucleotide is also useful because of theinteractions that exist between hormone-regulated pathways, stresspathways, nutritional pathways and development. Here, in addition topolynucleotides having similar transcription profiles and/or biologicalactivities, useful combinations include polynucleotides that may havedifferent transcription profiles but which participate in commonoverlapping pathways. The MA_diff Table(s) reports the transcript levelsof the experiment (see EXPT ID: 108562, 108563, 108519, 108520, 108521,108484, 108485, 108486). For transcripts that had higher levels in thesamples than the control, a “+” is shown. A “−” is shown for whentranscript levels were reduced in root tips as compared to the control.For more experimental detail see the Example section below.

GA genes are those sequences that showed differential expression ascompared to controls, namely those sequences identified in the MA_difftables with a “+” or “−” indication.

GA Genes Identified by Cluster Analyses of Differential Expression

GA Genes Identified by Correlation to Genes that are DifferentiallyExpressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of GA genes is any group in the MA_clust thatcomprises a cDNA ID that also appears in Expt ID 108562, 108563, 108519,108520, 108521, 108484, 108485, 108486 of the MA_diff table(s).

GA Genes Identified by Correlation to Genes that Cause PhysiologicalConsequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of GA genes.A group in the MA_clust is considered a GA pathway or network if thegroup comprises a cDNA ID that also appears in Knock-in or Knock-outtables that causes one or more of the phenotypes described in sectionabove.

GA Genes Identified by Amino Acid Sequence Similarity

GA genes from other plant species typically encode polypeptides thatshare amino acid similarity to the sequences encoded by corn andArabidopsis GA genes. Groups of GA genes are identified in the ProteinGroup table. In this table, any protein group that comprises a peptideID that corresponds to a cDNA ID member of a GA pathway or network is agroup of proteins that also exhibits GA functions/utilities.

Such GA responsive genes and gene products can function to eitherincrease or dampen the above phenotypes or activities either in responseto changes in GA concentration or in the absence of GA fluctuations.Further, promoters of GA responsive genes, as described in the Referencetables, for example, are useful to modulate transcription that isinduced by GA or any of the following phenotypes or biologicalactivities below.

III.C.5.a. Use of GA Responsive Genes to Modulate Phenotypes:

GA responsive genes and gene products are useful to or modulate one ormore phenotypes including growth, promotes root growth, promotes celldivision, promotes stem elongation, secondary (woody) growth, promotesgrowth in leaves, biomass, increase in stem and leaf mass, increase inxylem fiber length and biomass production, development, cell growth,fruit development, seed development, dormancy, breaks dormancy in seedsand buds, promotes trichome formation, decrease senescence, regulationof fertility, stress responses, and flowering time.

Further, any desired sequence can be transcribed in similar temporal,tissue, or environmentally specific patterns as the GA responsive geneswhen the desired sequence is operably linked to a promoter of a GAresponsive gene.

To regulate any of the phenotype(s) above, activities of one or more ofthe GA response genes or gene products can be modulated and tested byscreening for the desired trait. Specifically, the gene, mRNA levels, orprotein levels can be altered in a plant utilizing the proceduresdescribed herein and the phenotypes can be assayed. As an example, aplant can be transformed according to Bechtold and Pelletier (1998,Methods. Mol. Biol. 82:259-266) and visually inspected for the desiredphenotype or metabolically and/or functionally assayed according toHedden and Proebsting (1999, Plant Physiol. 119:365-370), Hedden andPhillips (1999, Current Opinion in Plant Biotech. 11:130-137), Perazzaet al (1998, Plant Physiol. 117:375-383), Kende and Zeevart (1997, PlantCell 9:1197-1210) and van der Knaap et al. (2000, Plant Physiol.122:695-704).

III.C.5.b. Use of GA-Responsive Genes to Modulate BiochemicalActivities:

The activities of one or more of the GA responsive genes can bemodulated to change biochemical or metabolic activities and/or pathwayssuch as those noted below. Such biological activities can be measuredaccording to the citations included in the Table below:

BIOCHEMICAL OR METABOLIC ACTIVITIES CITATIONS INCLUDING PROCESS AND/ORPATHWAYS ASSAYS Cell Growth Biosynthesis of Gas Hedden and Proebstingand Cell wall loosening and (1999, Plant Physiol. Differentiation cellexpansion 119: 365-370) GA deactivation Cosgrove (1993, New Major growthpromoting Phytol. 124: 1-23) metabolic pathways Hedden and Proebsting(1999, Plant Physiol. 119: 365-370) Perception Receptors Koornneef andvan der Veen and Signal (1980, TAG 58: 257-263) Transduction Synthesisof Bethke and Jones (1998, transcriptional regulators Curr. Opin. PlantBiol. Calcium and Calmodulin 1: 440-446)

Other biological activities that can be modulated by the GA responsivegenes and gene products are listed in the Reference Tables. Assays fordetecting such biological activities are described in the Protein Domaintable.

GA responsive genes are characteristically differentially transcribed inresponse to fluctuating GA levels or concentrations, whether internal orexternal to an organism or cell. The MA_diff table(s) report(s) thechanges in transcript levels of various GA responsive genes in entireseedlings at 1 and 6 hours after a plant was sprayed with a Hoagland'ssolution enriched with GA as compared to seedlings sprayed withHoagland's solution only.

The data from this time course can be used to identify a number of typesof GA responsive genes and gene products, including “early responders,”and “delayed responders.” Profiles of some GA responsive genes are shownin the Table below with examples of associated biological activities.

EXAMPLES OF TRANSCRIPT PHYSIOLOGICAL BIOCHEMICAL LEVELS TYPE OF GENESCONSEQUENCES ACTIVITY Up regulated Early responders to GA perceptionTranscription factors transcripts GA GA transport Transporters (level at1 hr ≈ 6 hr) Genes induced by Modulation of GA Change in cell (level at1 hr > 6 hr) GA response membrane structure transduction Kinases andpathways phosphatases Specific gene Transcription activatorstranscription Change in chromatin initiation structure and/or Growthstimulating localized DNA topology pathway induction Cell wall proteinsMetabolic Enzymes Up regulated Maintenance of GA Maintenance ofTranscription factors transcripts response response to GA Specificfactors (level at 1 hr < 6 hr) “Delayed” responders Induction of GA(initiation and metabolic pathways elongation) for protein synthesisMaintenance of mRNA stability Metabolic enzymes Down-regulated Earlyrepressor Negative regulation Transcription factors transcriptsresponders to GA of GA pathways Calmodulin (level at 1 hr ≈ 6 hr) Genesrepressed by released Change in protein (level at 6 hr > 1 hr) GAReduced activity of structure by phosphorylation Genes whose repressedpathways (kinases) or activities are dephosphoryaltion diminished or(phosphatases) mRNAs are unsTable Change in chromatin in the presence ofGA structure and/or DNA topology Down-regulated Delayed respondersMaintenance or GA Transcription factors transcripts Genes repressed byrepressed pathways Kinases and (level at 1 hr > 6 hr) GA phosphatasesGenes whose Stability factors for activities are protein translationdiminished or Metabolic enzymes mRNAs are unsTable in the presence of GA

Use of Promoters of GA Responsive Genes

Promoters of GA responsive genes are useful for transcription of anydesired polynucleotide or plant or non-plant origin. Further, anydesired sequence can be transcribed in a similar temporal, tissue, orenvironmentally specific patterns as the GA responsive genes where thedesired sequence is operably linked to a promoter of a GA responsivegene. The protein product of such a polynucleotide is usuallysynthesized in the same cells, in response to the same stimuli as theprotein product of the gene from which the promoter was derived. Suchpromoter are also useful to produce antisense mRNAs to down-regulate theproduct of proteins, or to produce sense mRNAs to down-regulate mRNAsvia sense suppression.

III.D. Metabolism Affecting Genes, Gene Components and Products

III.D.1. Nitrogen Responsive Genes, Gene Components and Products

Nitrogen is often the rate-limiting element in plant growth, and allfield crops have a fundamental dependence on exogenous nitrogen sources.Nitrogenous fertilizer which is usually supplied as ammonium nitrate,potassium nitrate, or urea, typically accounts for 40% of the costsassociated with crops, such as corn and wheat in intensive agriculture.Increased efficiency of nitrogen use by plants should enable theproduction of higher yields with existing fertilizer inputs and/orenable existing yields of crops to be obtained with lower fertilizerinput, or better yields on soils of poorer quality. Also, higher amountsof proteins in the crops could also be produced more cost-effectively.

Changes in nitrogen concentration in the surrounding environment or incontact with a plant results in modulation of the activities of manygenes and hence levels of gene products. Examples of such “nitrogenresponsive” genes and gene products with these properties are shown inthe Reference, Sequence, Protein Group, Protein Group Matrix tables,MA_diff, MA_clust, Knock-in and Knock-out tables. These genes and/orproducts are responsible for effects on traits such as plant vigor andseed yield. They were discovered and characterized from a much largerset by experiments designed to find genes whose mRNA products changed inresponse to changing levels of available nitrogen to plants.

Manipulation of one or more “nitrogen responsive” gene activities isuseful to modulate the biological activities and/or phenotypes listedbelow. “Nitrogen responsive” genes and gene products can act alone or incombination. Useful combinations include nitrogen responsive genesand/or gene products with similar transcription profiles, similarbiological activities, or members of the same or functionally relatedbiochemical pathways. Whole pathways or segments of pathways arecontrolled by transcription factor proteins and proteins controlling theactivity of signal transduction pathways. Therefore, manipulation of thelevels of such proteins is especially useful for altering phenotypes andbiochemical activities of plants. The MA_diff Table(s) reports thetranscript levels of the experiment (see EXPT ID: 108592, 108593,108588, 108589, 108590, 108591, 108532, 108548, 108549, 108550, 108551,108454, 108455, 108487, 108488, 108489, and Nitrogen (relating to SMD3787, SMD 3789)). For transcripts that had higher levels in the samplesthan the control, a “+” is shown. A “−” is shown for when transcriptlevels were reduced in root tips as compared to the control. For moreexperimental detail see the Example section below.

Nitrogen genes are those sequences that showed differential expressionas compared to controls, namely those sequences identified in theMA_diff tables with a “+” or “−” indication.

Nitrogen Genes Identified by Cluster Analyses of Differential Expression

Nitrogen Genes Identified by Correlation to Genes that areDifferentially Expressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of Nitrogen genes is any group in the MA_clust thatcomprises a cDNA ID that also appears in Expt ID 108592, 108593, 108588,108589, 108590, 108591, 108532, 108548, 108549, 108550, 108551, 108454,108455, 108487, 108488, 108489, and Nitrogen (relating to SMD 3787, SMD3789) of the MA_diff table(s).

Nitrogen Genes Identified by Correlation to Genes that CausePhysiological Consequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of Nitrogengenes. A group in the MA_clust is considered a Nitrogen pathway ornetwork if the group comprises a cDNA ID that also appears in Knock-inor Knock-out tables that causes one or more of the phenotypes describedin section above.

Nitrogen Genes Identified by Amino Acid Sequence Similarity

Nitrogen genes from other plant species typically encode polypeptidesthat share amino acid similarity to the sequences encoded by corn andArabidopsis Nitrogen genes. Groups of Nitrogen genes are identified inthe Protein Group table. In this table, any protein group that comprisesa peptide ID that corresponds to a cDNA ID member of a Nitrogen pathwayor network is a group of proteins that also exhibits Nitrogenfunctions/utilities.

Such “nitrogen responsive” genes and gene products can function eitherto either increase or dampen the phenotypes and activities below, eitherin response to changes in nitrogen concentration or in the absence ofnitrogen fluctuations.

Further, promoters of nitrogen responsive genes, as described in theReference tables, for example, are useful to modulate transcription thatis induced by nitrogen or any of the following phenotypes or biologicalactivities below.

III.D.5.a. Use of Nitrogen-Responsive Genes to Modulate Phenotypes

“Nitrogen responsive” genes and gene products can be used to alter ormodulate one or more phenotypes including plant development, initiationof the reproduction cycle from a vegetative state (such as flowerdevelopment time and time to fruit maturity); root development andinitiation (such as root branching, lateral root, initiation and/ordevelopment, nodule formation and nitrogen assimilation from anynitrogen-fixing symbions), growth rate, whole plant (including height,flowering time, etc.), organs (such as flowers, fruits, stems, leaves,roots, and lateral roots), biomass (such as fresh and dry weight duringany time in plant life, such as maturation); number, size, and weight offlowers; seeds; branches, and leaves); total plant nitrogen content,amino acid/protein content of whole plant or parts, seed yield (such asnumber, size, weight, harvest index and content and composition, e.g.,amino acid, nitrogen, oil, protein, and carbohydrate) and fruit yield(such as number, size, weight, harvest index, content and composition,e.g., amino acid, nitrogen, oil, protein, carbohydrate, and water.

To regulate any of the phenotype(s) above, activities of one or more ofthe nitrogen responsive genes or gene products can be modulated and theplants can be tested by screening for the desired trait. Specifically,the gene, mRNA levels, or protein levels can be altered in a plantutilizing the procedures described herein and the phenotypes can bescreened for variants as in Winkler et al. (1998) Plant Physiol 118:743-50 and assayed, for example, in accordance to Zhang (1999) Proc.Natl. Acad. Sci. 96(11): 6529-34; or Zhang and Forde (1998) Science279(5349):407-9; Scheible, W., Lauerer, M., Schultze, E.-D., Caboche,M., and Sitt, M. (1997). Plant J. 11, 671-691; Chevalier C, Bourgeois E,Just D, Raymond P. Plant J. 1996 January; 9(1):1-11.

III.D.5.b. Use of Nitrogen-Responsive Genes to Modulate BiochemicalActivities

The activities of one or more of the nitrogen responsive genes can bemodulated to change biochemical or metabolic activities and/or pathwayssuch as those noted below. Such biological activities are documented andcan be measured according to the citations included in the Table below:

Biochemical Or Metabolic Process Activities And/Or Pathways Citationsincluding assays Nitrate And Ammonium NO₃ ⁻ Influx And Efflux Lejay etal. (1999) Plant J. 18(5): Uptake and Assimilation 509-519 NitrateChannels Liu et al. (1999) Plant Cell 11: 865-874; and Wang et al.(1998)Proc. Natl. Acad. Sci. USA 95: 15134-15139 Changes In Membrane- Meharget al. (1995) J. Membr. Potential Biol. 145: 49-66; and Wang et al.(1998), supra Amino Acid Synthesis Glutamine Synthesis And Coruzzi etal. U.S. Pat. No. Then Biosynthesis Of Other 5,955,651; and Amino AcidsOliveira et al. (1999) Plant. Phys. 121: 301-309 Asparagine SynthesisAnd LAM ET AL. (1998) PLANT J. Then Biosynthesis Of Other 16(3): 345-353Amino Acids Coordination Of Carbon Light-Regulation Of Major Lam et al.(1998), supra; And Nitrogen Metabolism Central Carbon And Lejay et al.(1999), supra; and Nitrogen Metabolic Oliveira et al. (1999), supraPathways To Coordinate Growth Carbohydrate And Nitrogen Lam et al.(1998) supra; Control Of Carbohydrate Lejay et al. (1999) supra; and AndOrganic Nitrogen Oliveira et al. (1999) supra Accumulation PathwaysNitrogen Loading And Nitrogen Transport From Walker et al. (1999)210(1): 9-18 Unloading Source To Sinks Elsheikh et al. (1997) 51(2):137-44. Nitrogen Storage Accumulation Of Amino Johnson et al. (1990)Plant Cell Acids And/Or Storage 2(6): 525-32. Proteins In VacuolesHerman and Larkins (1999) Plant Cell. 11(4): 601-14. Ammonium PlastidAmmonium Crawford (1995) Plant Cell Detoxification Storage/Glutamine7(7): 859-68. Synthesis Zhang and Forde (1998) Science 279: 407-409.Cell Growth DIVISION AND/OR Zhang and Forde (1998) Science ELONGATION279: 407-409. Coruzzi et al. U.S. Pat. No. 5,955,651

Other biological activities that can be modulated by the nitrogenresponsive genes and their products are listed in the Reference tables.Assays for detecting such biological activities are described in theDomain section above.

Nitrogen responsive genes are characteristically differentiallytranscribed in response to fluctuating nitrogen levels orconcentrations, whether internal or external to an organism or cell. TheMA_diff table reports the changes in transcript levels of variousnitrogen responsive genes in the aerial parts of a seedling at 2, 6, 9and 12 hours after a plant was sprayed with a solution enriched withammonium nitrate as compared to seedlings sprayed with water. TheMA_diff reports the changes in transcript levels of various nitrogenresponsive genes in roots at 12 and 24 hours that were cut fromseedlings transferred from a high to low potassium nitrate environmentcompared to control seedlings transferred to a high potassium nitrateenvironment.

The data from this time course reveal a number of types of nitrogenresponsive genes and gene products, including “early responders,” and“delayed nitrogen responders”. Profiles of the individual categories ofnitrogen responsive genes are shown in the Tables below together withexamples of the kinds of associated biological activities that aremodulated when the activities of one or more such genes vary in plants.

Low to High Ammonium Nitrate Experiment

Functional Gene Expression Category Of Physiological Levels GeneConsequences Examples Of Gene Products Upregulated Early Perception OfTranscription Factors Transcripts Responders To Nitrogen Transporters(Level At 2 h ≅ 6, Nitrogen Induced Nitrogen Inhibitors Of Nitrogen 9 Or12 h) Or Uptake Into Cells Fixation (Level At 2 h > 6, Induction OfNitrogen Components Of Pathways 9 Or 12 h) Response TransductionReleased From Repression Pathways Transaminases Initiation Of SpecificAmino Acid Biosynthetic Gene Transcription Enzymes Upregulated DelayedMaintenance Of High Nitrogen Metabolic Transcripts Nitrogen NitrogenMetabolism Pathway Enzymes (Level At 2 h < 6, Responders And GrowthTransaminases 9, Or 12 h Amino Acid Biosynthetic Enzymes Factors InducedIn Coordination And Control Of Central Carbon And Nitrogen MetabolismCell Wall And Cell Growth-Promoting Pathway Enzymes Storage ProteinsDown Regulated Early Negative Regulation Transcription FactorsTranscripts Responder Of Nitrogen Utilization Kinases And Phosphatases(Level At 2 h ≅ 6, Repressors Of Pathways Released Cytoskeletal Proteins9 Or 12 h) Or Nitrogen Modulating Cell Structure (Level At 6, 9 OrUtilization Pathways Of C And N Chromatin Structure 12 h > 2 h) PathwaysMetabolism Required Regulatory Proteins Genes With At Lower LevelsMetabolic Enzymes Discontinued Decline In Presence Of TransportersExpression Or High Nitrogen Proteins And Rna UnsTable Mrna TurnoverSystems Following Nitrogen Uptake Level At 2 Hours > Delayed NegativeRegulation Transcription Factors 6, 9 Or 12 Hours Response Of NitrogenUtilization Kinases And Phosphatases Repressors Of Pathways ReleasedCytoskeletal Proteins Nitrogen Modulating Cell Structure UtilizationPathways Of C And N Chromatin Structure Pathways Metabolism RequiredRegulatory Proteins Genes With At Lower Levels Metabolic EnzymesDiscontinued Decline In Presence Of Transporters Expression Or HighNitrogen Protein And Rna Turnover UnsTable Mrna Systems FollowingNitrogen Uptake

High to Low Potassium Nitrate Experiment

Examples Of Biochemical Gene Expression Functional Type Of BiologicalActivities Of Gene Levels Category Of Gene Activity Products UpregulatedEarly Responders Perception Of Low Transcription Factors - Transcripts(Level To Low Nitrate Nitrate Controlling Transcription At 12 h~ 24 h)Nitrogen Uptake Into Transporters - Facilitating (Level At CellsTransport 12 h > 24 h) Low Nitrogen Signal Cell Wall/MembraneTransduction Response Structure Determining Pathways Proteins InitiationOf Specific Kinases And Gene Transcription Phosphatases- Initiation OfNitrogen Regulating Signal Fixation Transduction Pathways CytoskeletalProteins-Modulating Cell Structure Chromatin Structure And/Or DnaTopology Proteins Protein-Protein Interaction Participants MetabolicEnzymes- Nitrogen Turnover Enzymes And Pathway Components UpregulatedDelayed Low Maintenance Of Low Transcription Factors - TranscriptsNitrate Nitrogen Response Controlling Transcription (Level 12 h < 24 h)Responders Pathways (See the Table Transporters - Facilitating Above)Transport Cell Wall/Membrane Structure Determining Proteins Kinases AndPhosphatases- Regulating Signal Transduction Pathways CytoskeletalProteins-Modulating Cell Structure Chromatin Structure And/Or DnaTopology Proteins Protein-Protein Interaction Participants MetabolicEnzymes- Nitrogen Turnover Enzymes And Pathway Components Down-RegulatedEarly Repressor Negative Regulation Of Transcription Factors Transcripts(Level Responders To Low Nitrogen-Mediated Cell At 12 h~24 h) LowNitrate Pathways And/Or Wall/Membrane Structure (Level At Genes WhoseResponses Released Determining Proteins 12 h > 24 h) Expression IsPathways In C And N Factors For Discontinued Or Metabolism Required AtPromoting Protein Mrna Is UnsTable Lower Levels Decline In TranslationIn Presence Of The Presence Of Low Kinases And Low Nitrate NitratePhosphatases Cytoskeletal Proteins- Modulating Cell Structure ProteinAnd Rna Turnover Systems Down-Regulated Delayed Negative Regulation OfTranscription Factors Transcripts Repressor Low Nitrogen-Mediated Cell(Level At Responders To Pathways And/Or Wall/Membrane Structure 12 h <24 h) Low Nitrate Responses Released Determining Proteins Genes WhosePathways In C And N Factors For Expression Is Metabolism Required AtPromoting Protein Discontinued Or Lower Levels Decline In TranslationmRNA Is The Presence Of Low Kinases And UnsTable In Nitrate PhosphatasesPresence Of Low Cytoskeletal Proteins- Nitrate Modulating Cell StructureProtein And Rna Turnover Systems Chromatin Structure And/Or Dna TopologyProteins

Further, any desired sequence can be transcribed in similar temporal,tissue, or environmentally specific patterns as the nitrogen responsivegenes when the desired sequence is operably linked to a promoter of anitrogen responsive gene.

III.D.2. Circadian Rhythm (Clock) Responsive Genes, Gene Components andProducts

Often growth and yield are limited by the ability of a plant to toleratestress conditions, including water loss. To combat such conditions,plant cells deploy a battery of responses that are controlled by aninternal circadian clock, including the timed movement of cotyledons andleaves, timed movements in guard cells in stomata, and timed biochemicalactivities involved with sugar and nitrogen metabolism. These responsesdepend on the functioning of an internal circadian clock, that becomesentrained to the ambient light/dark cycle, and a series of downstreamsignaling events leading to transcription independent and transcriptiondependent stress responses.

A functioning circadian clock can anticipate dark/light transitions andprepare the physiology and biochemistry of a plant accordingly. Forexample, expression of a chlorophyll a/b binding protein (CAB) iselevated before daybreak, so that photosynthesis can operate maximallyas soon as there is light to drive it. Similar considerations apply tolight/dark transitions and to many areas of plant physiology such assugar metabolism, nitrogen metabolism, water uptake and water loss,flowering and flower opening, epinasty, germination, perception ofseason, and senescence.

Manipulation of one or more clock gene activities is useful to modulatethe biological processes and/or phenotypes listed below. Clockresponsive genes and gene products can act alone or in combination.Useful combinations include clock responsive genes and/or gene productswith similar transcription profiles, similar biological activities, ormembers of the same or functionally related biochemical pathways. Wholepathways or segments of pathways are controlled by transcription factorproteins and proteins controlling the activity of signal transductionpathways. Therefore, manipulation of such protein levels is especiallyuseful for altering phenotypes and biochemical activities of plants. TheMA_diff Table(s) reports the transcript levels of the experiment (seeEXPT ID: Circadian (relating to SMD 2344, SMD 2359, SMD 2361, SMD 2362,SMD 2363, SMD 2364, SMD 2365, SMD 2366, SMD 2367, SMD 2368, SMD 3242)).For transcripts that had higher levels in the samples than the control,a “+” is shown. A “−” is shown for when transcript levels were reducedin root tips as compared to the control. For more experimental detailsee the Example section below.

Circadian genes are those sequences that showed differential expressionas compared to controls, namely those sequences identified in theMA_diff tables with a “+” or “−” indication.

Circadian Genes Identified by Cluster Analyses of DifferentialExpression

Circadian Genes Identified by Correlation to Genes that areDifferentially Expressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork;

A pathway or network of Circadian genes is any group in the MA_clustthat comprises a cDNA ID that also appears in Expt ID Circadian(relating to SMD 2344, SMD 2359, SMD 2361, SMD 2362, SMD 2363, SMD 2364,SMD 2365, SMD 2366, SMD 2367, SMD 2368, SMD 3242) of the MA_difftable(s).

Circadian Genes Identified by Correlation to Genes that CausePhysiological Consequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of Circadiangenes. A group in the MA_clust is considered a Circadian pathway ornetwork if the group comprises a cDNA ID that also appears in Knock-inor Knock-out tables that causes one or more of the phenotypes describedin section above.

Circadian Genes Identified by Amino Acid Sequence Similarity

Circadian genes from other plant species typically encode polypeptidesthat share amino acid similarity to the sequences encoded by corn andArabidopsis Circadian genes. Groups of Circadian genes are identified inthe Protein Group table. In this table, any protein group that comprisesa peptide ID that corresponds to a cDNA ID member of a Circadian pathwayor network is a group of proteins that also exhibits Circadianfunctions/utilities.

Such clock responsive genes and gene products can function to eitherincrease or dampen the above phenotypes or activities either in responseto changes in daylength or in response to changes in light quality.Further, promoters of cirdadian (clock) responsive genes, as describedin the Reference tables, for example, are useful to modulatetranscription that is induced by circadian or any of the followingphenotypes or biological activities below. Further, any desired sequencecan be transcribed in similar temporal, tissue, or environmentallyspecific patterns as the circadian (clock) responsive genes when thedesired sequence is operably linked to a promoter of a circadian (clock)responsive gene.

The expression of many genes is modulated by the clock. Microarraytechnology allows monitoring of gene expression levels for thousands ofgenes in a single experiment. This is achieved by hybridizing labeledfluorescent cDNA pools to glass slides that contain spots of DNA (Schenaet al. (1995) Science 270: 467-70). The US Arabidopsis FunctionalGenomics Consortium (AFGC) has recently made public the results fromsuch microarray experiments conducted with AFGC chips containing some10,000 non-redundant ESTs, selected from about 37,000 randomly sequencedESTs generated from mRNA of different tissues and developmental stages.

The sequences of the ESTs showing at least two-fold increases ordecreases in response to the circadian rhythm clock at various timesthrough the 24 hour cycle relative to the controls were identified,compared to the Ceres full length cDNA and genomic sequence databanks,and equivalent Ceres clones identified. The MA_diff table reports theresults of this analysis, indicating those Ceres clones which are up ordown regulated over controls, thereby indicating the Ceres clones whichrepresent clock responsive genes.

III.D.2.a. Use of Circadian Rhythm (Clock) Responsive Genes to ModulatePhenotypes

Clock responsive genes and gene products are useful to or modulate oneor more phenotypes including timing phenotypes, dormancy, germination,cotyledon opening, first leaves, juvenile to adult transition, bolting,flowering, pollination, fertilization, seed development, seed set, fruitdrop, senescence, epinasty, biomass, fresh and dry weight during anytime in plant life, such as maturation, number of flowers, seeds,branches, and/or leaves, seed yield, including number, size, weight,and/or harvest index, fruit yield, including number, size, weight,and/or harvest index, plant development, time to fruit maturity, cellwall strengthening and reinforcement, stress tolerance, droughttolerance, flooding tolerance, and uv tolerance

To regulate any of the phenotype(s) above, activities of one or more ofthe clock responsive genes or gene products can be modulated and theplants can be tested by screening for the desired trait. Specifically,the gene, mRNA levels, or protein levels can be altered in a plantutilizing the procedures described herein and the phenotypes can bescreened for variants as in Anderson et al. (1997) Plant Cell 9:1727-1743; Heintzen et al. (1997) Proc. Natl. Acad. Sci. USA 94:8515-20; Schaffer et al. (1998) Cell 93:1219-1229; Somers et al. (1998)Development 125: 485-494; Somers et al. (1998) Science 282: 1488-1490;Wang and Tobin (1998) Cell 93: 1207-1217; Zhong et al. (1998) Plant Cell10: 2005-2017; Sugano et al. (1998) Proc. Natl. Acad. Sci. USA 95:11020-11025; Dowson-Day and Millar (1999) Plant J 17: 63-71; Green andTobin (1999) Proc. Natl. Acad. Sci. USA 96: 4176-419; Staiger and Apel(1999) Mol. Gen. Genet. 261: 811-819; Strayer and Kay (1999) Curr. Opin.Plant Biol. 2:114-120; Strayer et. al. (2000) Science 289:768-771; Krepset al. (2000) J Biol Rhythms (2000) 15:208-217; Nelson et al. (2000)Cell 101:331-340; Somers et al. (2000) Cell 101:319-329.

III.D.2.b. Use of Active Clock Responsive Genes to Modulate BiochemicalActivities

The activities of one or more of the clock responsive genes can bemodulated to change biochemical or metabolic activities and/or pathwayssuch as those noted below. Such biological activities are documented andcan be measured according to the citations above and included in theTable below:

BIOCHEMICAL OR METABOLIC ACTIVITIES CITATIONS INCLUDING AND/OR PATHWAYSASSAYS PROCESS Germination and seedling Cold, light and water modulatedBognar et al. (1999) Proc. development signal transduction pathways,Natl. Acad. Sci. USA receptors, kinases, PAS domain 96: 14652-14657;Sugano et al (1999) Proc. Natl. Acad. Sci. USA 96: 12362-12366;Dowson-Day and Millar (1999) Plant J 17: 63-71; Somers et al. (2000)Cell 101: 319-329; Zhong et al. (1998) Plant Cell 10: 2005-2017 Growthtransitions and Cold and light modulated signal Nelson et al. (2000)Cell flowering transduction pathways, 101: 331-340; Fowler et al.receptors, kinases, PAS domain (1999) EMBO J. 18: 4679-4688 Tuberformation Cold and light modulated signal Yanovsky et al. (2000) Planttransduction pathways J. 23: 223-232 METABOLISM Lipid metabolismMembrane lipid synthesis Bradley and Reddy (1997) J. including omega-3fatty acid Bacteriol. 179: 4407-4410; desaturase, lipases, lipid Martin,M et al. 1999 Europe transfer proteins J. Biochem 262: 283-290 Sugarmetabolism Glycosylhydrolases, Liu et al. (1996) Plantglycosyltransferases, amylases, Physiol. 112: 43-51; Millar sucrosesynthase, CAB, and Kay (1996) Proc Natl Rubisco, light signal Acad SciUSA 93: 15491-15496; transduction Wang et al. (1997) Plant Cell 9:491-507; Shinohara et al (1999) J. Biol. Chem. 273: 446-452 Nitrogenmetabolism Aminotransferases, arginase, Bradley and Reddy (1997) J.proteases and vegetative storage Bacteriol. 179: 4407-4410 proteins,aromatic amino acid synthesis Photorespiration Mitochondrial,chloroplast and Zhong and McClung (1996) peroxisomal photorespiratoryMol. Gen. Genet. 251: 196-203; enzymes, serine hydroxymethyl McClung(1997) Free. transferases, catalase Radic. Biol. Med. 23: 489-496;McClung et al. (2000) Plant Physiol. 123: 381-392 Responses toEnvironmental Expression of genes involved in McClung (1997) Free RadicStress responses to drought, salt, UV Biol Med 23: 489-496; Shi et al.(2000) Proc. Natl. Acad. Sci. USA 97: 6896-6901

Other biological activities that can be modulated by the clockresponsive genes and their products are listed in the Reference tables.Assays for detecting such biological activities are described in theProtein Domain table.

Clock responsive genes are characteristically differentially transcribedin response to fluctuations in an entrained oscillator, which isinternal to an organism and cell. The MA_diff table(s) report(s) thechanges in transcript levels of various clock responsive genes in aplant.

Profiles of clock responsive genes are shown in the table below withexamples of which associated biological activities are modulated whenthe activities of one or more such genes vary in plants.

EXAMPLES OF PHYSIOLOGICAL BIOCHEMICAL TRANSCRIPT LEVELS TYPE OF GENESCONSEQUENCES ACTIVITY Up regulated Responders to Circadian rhythmMetabolic enzymes transcripts circadian rhythm perception Genes inducedby Metabolisms Change in cell rythm affected by membrane structureCircadian rhythm and potential Synthesis of Kinases and secondaryphosphatases metabolites Transcription and/or proteins activatorsModulation of Change in clock response chromatin structure transductionand/or localized pathways DNA topology Specific gene Enzymes in lipid,transcription sugar and nitrogen initiation metabolism Enzymes inphotorespiration and photosynthesis Down-regulated Responders toNegative Transcription transcripts circadian rhythm. regulation offactors Repressors of circadian Change in protein circadian “state” ofpathways released structure by metabolism Changes in phosphorylationGenes repressed by pathways and (kinases) or rhythm processesdephosphoryaltion Genes with operating in cells (phosphatases)discontinued Changes in Change in expression or metabolism otherchromatin structure unsTable mRNA in than circadian and/or DNA presenceof zinc pathways topology Stability of factors for protein synthesis anddegradation Metabolic enzymes in light, sugar, lipid and nitrogenmetabolism

Use of Promoters of Clock Responsive Genes

Promoters of Clock responsive genes are useful for transcription of anydesired polynucleotide or plant or non-plant origin. Further, anydesired sequence can be transcribed in a similar temporal, tissue, orenvironmentally specific patterns as the Clock responsive genes wherethe desired sequence is operably linked to a promoter of a Clockresponsive gene. The protein product of such a polynucleotide is usuallysynthesized in the same cells, in response to the same stimuli as theprotein product of the gene from which the promoter was derived. Suchpromoter are also useful to produce antisense mRNAs to down-regulate theproduct of proteins, or to produce sense mRNAs to down-regulate mRNAsvia sense suppression.

III.D.3. Blue Light (Phototropism) Responsive Genes, Gene Components andProducts

Phototropism is the orientation or growth of a cell, an organism or partof an organism in relation to a source of light. Plants can sense red(R), far-red (FR) and blue light in their environment and responddifferently to particular ratios of these. For example, a low R:FR ratioenhances cell elongation and favors flowering over leaf production, butblue light regulated cryptochromes also appear to be involved indetermining hypocotyl growth and flowering time.

Phototropism of Arabidopsis thaliana seedlings in response to a bluelight source is initiated by nonphototropic hypocotyl 1 (NPH1), a bluelight-activated serine-threonine protein kinase, but the downstreamsignaling events are not entirely known. Blue light treatment leads tochanges in gene expression. These genes have been identified bycomparing the levels of mRNAs of individual genes in dark-grownseedlings, compared with in dark grown seedlings treated with 1 hour ofblue light. Auxin also affects blue light phototropism. The effect ofAuxin on gene expression stimulated by blue light has been explored bystudying mRNA levels in a mutant of Arabidopsis thaliana nph4-2, grownin the dark and, treated with blue light for 1 hour compared with wildtype seedlings treated similarly. This mutant is disrupted forAuxin-related growth and Auxin-induced gene transcription. Geneexpression was studied using microarray technology.

Microarray technology allows monitoring of gene expression levels forthousands of genes in a single experiment. This is achieved byhybridizing labeled fluorescent cDNA pools to glass slides that containspots of DNA (Schena et al. (1995) Science 270: 467-70). The USArabidopsis Functional Genomics Consortium (AFGC) has recently madepublic the results from such microarray experiments conducted with AFGCchips containing some 10,000 non-redundant ESTs, selected from about37,000 randomly sequenced ESTs generated from mRNA of different tissuesand developmental stages.

The sequences of the ESTs showing at least two-fold increases ordecreases over the controls were identified, compared to the Ceresfull-length cDNA and genomic sequence databanks, and the equivalentCeres clones identified. The MA_diff table(s) report(s) the results ofthis analysis, indicating those Ceres clones which are up or downregulated over controls, thereby indicating the Ceres clones whichrepresent blue light responsive genes and of those which are blue lightresponsive in the absence of nph4 gene activity. The MA_diff Table(s)reports the transcript levels of the experiment (see EXPT ID:Phototropism (relating to SMD 4188, SMD 6617, SMD 6619)). Fortranscripts that had higher levels in the samples than the control, a“+” is shown. A “−” is shown for when transcript levels were reduced inroot tips as compared to the control. For more experimental detail seethe Example section below.

Blue Light genes are those sequences that showed differential expressionas compared to controls, namely those sequences identified in theMA_diff tables with a “+” or “−” indication.

Blue Light Genes Identified by Cluster Analyses of DifferentialExpression

Blue Light Genes Identified by Correlation to Genes that areDifferentially Expressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of Blue Light genes is any group in the MA_clustthat comprises a cDNA ID that also appears in Expt ID Phototropism(relating to SMD 4188, SMD 6617, SMD 6619) of the MA_diff table(s).

Blue Light Genes Identified by Correlation to Genes that CausePhysiological Consequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of BlueLight genes. A group in the MA_clust is considered a Blue Light pathwayor network if the group comprises a cDNA ID that also appears inKnock-in or Knock-out tables that causes one or more of the phenotypesdescribed in section above.

Blue Light Genes Identified by Amino Acid Sequence Similarity

Blue Light genes from other plant species typically encode polypeptidesthat share amino acid similarity to the sequences encoded by corn andArabidopsis Blue Light genes. Groups of Blue Light genes are identifiedin the Protein Group table. In this table, any protein group thatcomprises a peptide ID that corresponds to a cDNA ID member of a BlueLight pathway or network is a group of proteins that also exhibits BlueLight functions/utilities.

III.D.3.a. Use of Blue Light Responsive Genes, Gene Components andProducts to Modulate Phenotypes

Changes in blue light in a plant's surrounding environment result inmodulation of many genes and gene products. Examples of such blue lightresponse genes and gene products are shown in the REFERENCE and SEQUENCETables. These genes and/or products are responsible for effects ontraits such as plant vigor and seed yield.

While blue light responsive polynucleotides and gene products can actalone, combinations of these polynucleotides also affect growth anddevelopment. Useful combinations include different blue light responsivepolynucleotides and/or gene products that have similar transcriptionprofiles or similar biological activities, and members of the same orsimilar biochemical pathways. Whole pathways or segments of pathways arecontrolled by transcription factor proteins and proteins controlling theactivity of signal transduction pathways. Therefore, manipulation ofsuch protein levels is especially useful for altering phenotypes andbiochemical activities of plants. In addition, the combination of a bluelight responsive polynucleotides and/or gene product with otherenvironmentally responsive polynucleotide is also useful because of theinteractions that exist between hormone-regulated pathways, stress andpathogen induced pathways, nutritional pathways and development. Here,in addition to polynucleotides having similar transcription profilesand/or biological activities, useful combinations includepolynucleotides that may have different transcription profiles but whichparticipate in common or overlapping pathways.

III.D.3.b. Use of Blue Light Responsive Genes, Gene Components andProducts to Modulate Phenotypes

Blue light responsive genes and gene products can function to eitherincrease or dampen the above phenotypes or activities either in responseto changes in blue light response concentration or in the absence ofblue light responsive fluctuations. Further, promoters of blue lightresponsive genes, as described in the Reference tables, for example, areuseful to modulate transcription that is induced by blue light or any ofthe following phenotypes or biological activities below. Further, anydesired sequence can be transcribed in similar temporal, tissue, orenvironmentally specific patterns as the blue light responsive geneswhen the desired sequence is operably linked to a promoter of a bluelight responsive gene.

Blue light responsive genes and gene products can be used to alter ormodulate one or more phenotypes including growth, roots (elongation orgravitropism) and stems (such as elongation), development of cell (suchas growth or elongation), flower (including flowering time), seedling(including elongation), plant yield, and seed and fruit yield.

To regulate any of the phenotype(s) above, activities of one or more ofthe blue light responsive genes or gene products can be modulated andthe plants tested by screening for the desired trait. Specifically, thegene, mRNA levels, or protein levels can be altered in a plant utilizingthe procedures described herein and the phenotypes can be assayed. As anexample, a plant can be transformed according to Bechtold and Pelletier(1998, Methods. Mol. Biol. 82:259-266) and/or screened for variants asin Winkler et al. (1998) Plant Physiol 118: 743-50 and visuallyinspected for the desired phenotype or metabolically and/or functionallyassayed according to Liscum and Briggs (1995, Plant Cell 7: 473-85),Vitha et al. (2000, Plant Physiol 122: 453-61), Stowe-Evance et al.(1998, Plant Physiol 118: 1265-75), Baum et al. (1999, PNAS USA 96:13554-9), Huala et al. (1997) Science 278: 2120-2123), Kanegae et al.(2000, Plant Cell Physiol 41: 415-23), Khanna et al. (1999, Plant MolBiol 39: 231-42), Sakai et al. (2000, Plant Cell 12: 225-36), Parks etal (1996, Plant Physiol 110: 155-62) and Janoudi et al. (1997, PlantPhysiol 113: 975-79).

III.D.3.c. Use of Blue Light Responsive Genes, Gene Components andProducts to Modulate Biochemical Activities

The activities of one or more of the blue light responsive genes can bemodulated to change biochemical or metabolic activities and/or pathwayssuch as those noted below. Such biological activities can be measuredaccording to the citations included in the Table below:

BIOCHEMICAL OR METABOLIC ACTIVITIES AND/OR CITATIONS INCLUDING PROCESSPATHWAYS ASSAYS Cell Growth Cell Elongation Liscum and Briggs (1995)Plant and Seedling Cell 7: 473-85 Development Stem Root Vitha et al.(2000) Plant Physiol 122: 453-61 Signalling UV light Liscum and Briggs(1996) Plant Perception Physiol 112: 291-96 Far-red/Red light Parks etal. (1996) Plant Physiol Perception 110: 155-62 Phosphorylation ofLiscum and Briggs (1996) Plant cellular and nuclear- Physiol 112: 291-96localized proteins Activation and Synthesis Sakae et al. (2000) Plant ofTranscription Factors Cell 12: 225-36 Ca+2 levels Baum et al. (1999)PNAS USA 96: 13554-9 Pu and Robinson (1998) J Cell Sci 111: 3197-3207Auxin Concentration Estelle (1998) Plant Cell 10: 1775-8 Reed et al.(1998) Plant Physiol 118: 1369-78 Inter-photoreceptors Janoudi et al.(1997) Plant Physiol 113: 975-79

Other biological activities that can be modulated by blue light responsegenes and their products are listed in the REF Tables. Assays fordetecting such biological activities are described in the Domain sectionof the REF Table.

The specific genes modulated by blue light, in wild type seedlings andin the mutant deficient in transmitting Auxin effects are given in theReference and Sequence Tables. The kinds of genes discovered and some oftheir associated effects are given in the Table below.

EXAMPLES OF TRANSCRIPT PHYSIOLOGICAL BIOCHEMICAL LEVELS TYPE OF GENESCONSEQUENCES ACTIVITY Up regulated Responders to no Blue lightTransporters transcripts blue light in wild type perception Metabolicenzymes or to blue light in Metabolism Change in cell mutant lackingAuxin affected by blue membrane structure effects light and potentialSynthesis of Kinases and secondary phosphatases metabolites and/orTranscription proteins activators Modulation of blue Change in chromatinlight transduction structure and/or pathways localized DNA Specific genetopology transcription initiation Down-regulated Responders to no Bluelight Transcription factors transcripts blue light in wild typeperception Change in protein or to blue light in Metabolism structure bymutants lacking affected by blue phosphorylation Auxin effects light(kinases) or Genes with Synthesis of dephosphorylation discontinuedsecondary (phosphatases) expression or metabolites and/or Change inchromatin unsTable mRNA proteins structure and/or during responseModulation of blue DNA topology light transduction Stability factors forpathways protein synthesis and Specific gene degradation transcriptionMetabolic enzymes initiation Changes in pathways and processes operatingin cells Changes in metabolic pathways other than phototropic blue lightresponsive pathways

Use of Promoters of Blue Light Responsive Genes

Promoters of Blue Light responsive genes are useful for transcription ofany desired polynucleotide or plant or non-plant origin. Further, anydesired sequence can be transcribed in a similar temporal, tissue, orenvironmentally specific patterns as the Blue Light responsive geneswhere the desired sequence is operably linked to a promoter of a BlueLight responsive gene. The protein product of such a polynucleotide isusually synthesized in the same cells, in response to the same stimulias the protein product of the gene from which the promoter was derived.Such promoter are also useful to produce antisense mRNAs todown-regulate the product of proteins, or to produce sense mRNAs todown-regulate mRNAs via sense suppression.

III.D.4 Responsive Genes, Gene Components and Products

There has been a recent and significant increase in the level ofatmospheric carbon dioxide. This rise in level is projected to continueover the next 50 years. The effects of the increased level of carbondioxide on vegetation are just now being examined, generally in largescale, whole plant (often trees) experiments. Some researchers haveinitiated physiological experiments in attempts to define thebiochemical pathways that are either affected by and/or are activated toallow the plant to avert damage from the elevated carbon dioxide levels.A genomics approach to this issue, using a model plant system, allowsidentification of those pathways affected by and/or as having a role inaverting damage due to the elevated carbon dioxide levels and affectinggrowth. Higher agronomic yields can be obtained for some crops grown inelevated CO₂.

Microarray technology allows monitoring of gene expression levels forthousands of genes in a single experiment. This is achieved byhybridizing labeled fluorescent cDNA pools to glass slides that containspots of DNA (Schena et al. (1995) Science 270: 467-70). The U.S.Arabidopsis Functional Genomics Consortium (AFGC) has recently madepublic the results from such microarray experiments conducted with AFGCchips containing about 10,000 non-redundant ESTs, selected from about37,000 randomly sequenced ESTs generated from mRNA of different tissuesand developmental stages.

The sequences of the ESTs showing at least two-fold increases ordecreases in plants grown in higher CO₂ levels compared with plantsgrown at more normal CO₂ levels, were compared to the Ceres full lengthcDNA and genomic sequence databanks, and equivalent Ceres clones wereidentified. The MA_diff table reports the results of this analysis,indicating those Ceres clones which are up or down regulated overcontrols, thereby indicating the Ceres clones cDNA sequences that changein response to CO₂.

Examples of CO₂ responsive genes and gene products are shown in theReference, Sequence, Protein Group, Protein Group Matrix tables, MA_diffand MA_clust tables. While CO₂ responsive polynucleotides and geneproducts can act alone, combinations of these polynucleotides alsoaffect growth and development. Useful combinations include different CO₂responsive polynucleotides and/or gene products that have similartranscription profiles or similar biological activities, and members ofthe same or similar biochemical pathways. Whole pathways or segments ofpathways are controlled by transcription factor proteins and proteinscontrolling the activity of signal transduction pathways. Therefore,manipulation of such protein levels is especially useful for alteringphenotypes and biochemical activities of plants.

Manipulation of one or more CO₂ responsive gene activities is useful tomodulate the biological processes and/or phenotypes listed below. CO₂responsive genes and gene products can act alone or in combination.Useful combinations include genes and/or gene products with similartranscription profiles, similar biological activities, or members of thesame or functionally related biochemical pathways. Here, in addition topolynucleotides having similar transcription profiles and/or biologicalactivities, useful combinations include polynucleotides that may havedifferent transcription profiles but which participate in common oroverlapping pathways.

CO₂ responsive genes and gene products can function to either increaseor dampen the above phenotypes or activities. Further, promoters of CO₂responsive genes, as described in the Reference tables, for example, areuseful to modulate transcription that is induced by CO₂ or any of thefollowing phenotypes or biological activities below. Further, anydesired sequence can be transcribed in similar temporal, tissue, orenvironmentally specific patterns as the CO₂ responsive genes when thedesired sequence is operably linked to a promoter of a CO₂ responsivegene. The MA_diff Table(s) reports the transcript levels of theexperiment (see EXPT ID: CO2 (relating to SMD 7561, SMD 7562, SMD 7261,SMD 7263, SMD 3710, SMD 4649, SMD 4650)). For transcripts that hadhigher levels in the samples than the control, a “+” is shown. A “−” isshown for when transcript levels were reduced in root tips as comparedto the control. For more experimental detail see the Example sectionbelow.

CO2 genes are those sequences that showed differential expression ascompared to controls, namely those sequences identified in the MA_difftables with a “+” or “−” indication.

CO2 Genes Identified by Cluster Analyses of Differential Expression

CO2 Genes Identified by Correlation to Genes that are DifferentiallyExpressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of CO2 genes is any group in the MA_clust thatcomprises a cDNA ID that also appears in Expt ID CO2 (relating to SMD7561, SMD 7562, SMD 7261, SMD 7263, SMD 3710, SMD 4649, SMD 4650) of theMA_diff table(s).

CO2 Genes Identified by Correlation to Genes that Cause PhysiologicalConsequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of CO2genes. A group in the MA_clust is considered a CO2 pathway or network ifthe group comprises a cDNA ID that also appears in Knock-in or Knock-outtables that causes one or more of the phenotypes described in sectionabove.

CO2 Genes Identified by Amino Acid Sequence Similarity

CO2 genes from other plant species typically encode polypeptides thatshare amino acid similarity to the sequences encoded by corn andArabidopsis CO2 genes. Groups of CO2 genes are identified in the ProteinGroup table. In this table, any protein group that comprises a peptideID that corresponds to a cDNA ID member of a CO2 pathway or network is agroup of proteins that also exhibits CO2 functions/utilities.

III.D.4.a. Use of Co2 Responsive Genes to Modulate Phenotypes

CO₂ responsive genes and gene products are useful to or modulate one ormore phenotypes including catabolism, energy generation, atp, etc.,metabolism, carbohydrate synthesis, growth rate, whole plant, includingheight, flowering time, etc., organs, flowers, fruits, stems, leaves,roots, lateral roots, biomass, fresh and dry weight during any time inplant life, such as maturation; number, size, and weight of flowers;seeds; branches; leaves; total plant nitrogen content, aminoacid/protein content of whole plant or parts, seed yield (such asnumber, size, weight, harvest index, and content and composition, e.g.,amino acid, nitrogen, oil, protein, and carbohydrate); fruit yield;number, size, weight, harvest index; content and composition, e.g.,amino acid, nitrogen, oil, protein, carbohydrate, water; andphotosynthesis (such as carbon dioxide fixation).

To improve any of the phenotype(s) above, activities of one or more ofthe CO₂ responsive genes or gene products can be modulated and theplants tested by screening for the desired trait. Specifically, thegene, mRNA levels, or protein levels can be altered in a plant utilizingthe procedures described herein and the phenotypes can be assayed. As anexample, a plant can be transformed according to Bechtold and Pelletier(1998, Methods. Mol. Biol. 82:259-266) and/or screened for variants asin Winkler et al. (1998) Plant Physiol 118: 743-50 and visuallyinspected for the desired phenotype or metabolically and/or functionallyassayed according to Saito et al. (1994, Plant Physiol. 106: 887-95),Takahashi et al (1997, Proc. Natl. Acad. Sci. USA 94: 11102-07) andKoprivova et al. (2000, Plant Physiol. 122: 737-46).

III.D.2. Use of CO₂ Responsive Genes to Modulate Biochemical Activities

The activities of one or more of the CO₂ responsive genes can bemodulated to change biochemical or metabolic activities and/or pathwayssuch as those noted below. Such biological activities can be measuredaccording to the citations included in the Table below:

BIOCHEMICAL OR METABOLIC GENERAL ACTIVITIES AND/OR CITATIONS INCLUDINGCATEGORY PATHWAYS ASSAYS Cell Division Cell Cycle Control Genes Masle(2000) Plant Physiol. 122: 1399-1415 Starch Starch Biosynthesis Ludewiget al., (1998) Biosynthesis Enzymes And Pathways FEBS Lett. 429: 147-151Photosynthesis Photosynthetic Enzymes, Cheng et al., (1998) Plant e.g.,Rubisco Physiol 166: 715-723 Respiration Energy Metabolism Musgrave etal., (1986) Pathways Proc. Natl. Acad. Sci. USA 83: 8157-8161 CO₂ UptakeGuard Cell Stomata Allen et al., Plant Cell Control Systems (1999)11(9): 1785-1798 Ichida et al., Plant Cell (1997) 9(10): 1843-1857Hedrich et al., EMBO J (1993) 12(3): 897-901 CoordinationLight-Regulation Of Major Lam et al. (1998) Plant J. Of Carbon CentralCarbon And 16(3): 345-353 And Nitrogen Nitrogen Metabolic Lejay et al.(1999) Plant J. Metabolism Pathways To Coordinate 18(5): 509-519; andGrowth Oliveira et al. (1999) Plant. Phys. 121: 301-309 Carbohydrate AndLam et al. (1998) supra; Nitrogen Control Of Lejay et al. (1999) supra;Carbohydrate And Organic and Nitrogen Accumulation Oliveira et al.(1999) supra Pathways

Other biological activities that can be modulated by the CO₂ responsivegenes and gene products are listed in the Reference tables. Assays fordetecting such biological activities are described in the Protein Domaintable.

CO₂ responsive genes are characteristically differentially transcribedin response to fluctuating CO₂ levels or concentrations, whetherinternal or external to an organism or cell. The MA_diff tables reportthe changes in transcript levels of various CO₂ responsive genes thatare differentially expressed in response to high CO₂ levels.

Profiles of these different CO₂ responsive genes are shown in the Tablebelow with examples of associated biological activities.

EXAMPLES TRANSCRIPT TYPE OF PHYSIOLOGICAL OF BIOCHEMICAL LEVELS GENESCONSEQUENCES ACTIVITY Up Regulated Responders Changes In Generation OfTransporters Transcripts To Higher ATP Catabolic And Levels Of ChangesIn Catabolism Anabolic Enzymes CO₂ And Anabolism Enzymes and Change InCell Genes Pathways Membrane Structure Induced By Activation Of KrebsCycle And Potential CO₂ Specific Gene Kinases And TranscriptionInitiation Phosphatases Changes In Carbohydrate Transcription SynthesisActivators And Changes In Chloroplast Repressors Structure Change InChanges In Photosynthesis Chromatin Structure Changes In RespirationAnd/Or Localized DNA Topology Redox Control Down- Responders Changes InPathways And Transcription Regulated To Higher Processes Operating InFactors Transcripts Levels Of Cells Change In Protein CO₂ Changes InCatabolism and Structure By Genes Anabolism Phosphorylation Repressed ByChanges in Chloroplast (Kinases) Or CO₂ Structure Dephosphorylation(Phosphatases) Change In Chromatin Structure And/Or DNA TopologyStability Of Factors For Protein Synthesis And Degradation MetabolicEnzymes

Use of Promoters of CO2 Responsive Genes

Promoters of CO2 responsive genes are useful for transcription of anydesired polynucleotide or plant or non-plant origin. Further, anydesired sequence can be transcribed in a similar temporal, tissue, orenvironmentally specific patterns as the CO2 responsive genes where thedesired sequence is operably linked to a promoter of a CO2 responsivegene. The protein product of such a polynucleotide is usuallysynthesized in the same cells, in response to the same stimuli as theprotein product of the gene from which the promoter was derived. Suchpromoter are also useful to produce antisense mRNAs to down-regulate theproduct of proteins, or to produce sense mRNAs to down-regulate mRNAsvia sense suppression.

III.D.5. Mitochondria Electron Transport (Respiration) Genes, GeneComponents and Products

One means to alter flux through metabolic pathways is to alter thelevels of proteins in the pathways. Plant mitochondria contain manyproteins involved in various metabolic processes, including the TCAcycle, respiration, and photorespiration and particularly the electrontransport chain (mtETC). Most mtETC complexes consist ofnuclearly-encoded mitochondrial proteins (NEMPs) andmitochondrially-encoded mitochondrial proteins (MEMPs). NEMPs areproduced in coordination with MEMPs of the same complex and pathway andwith other proteins in multi-organelle pathways. Enzymes involved inphotorespiration, for example, are located in chloroplasts,mitochondria, and peroxisomes and many of the proteins arenuclearly-encoded. Manipulation of the coordination of protein levelswithin and between organelles can have critical and global consequencesto the growth and yield of a plant. Genes which are manipulated byinterfering with the mtETC have been characterized using microarraytechnology.

Microarray technology allows monitoring of gene expression levels forthousands of genes in a single experiment. This is achieved byhybridizing labeled fluorescent cDNA pools to glass slides that containspots of DNA (Schena et al. (1995) Science 270: 467-70). The USArabidopsis Functional Genomics Consortium (AFGC) has recently madepublic the results from such microarray experiments conducted with AFGCchips containing about 10,000 non-redundant ESTs, selected from about37,000 randomly sequenced ESTs generated from mRNA of different tissuesand developmental stages.

The sequences of the ESTs showing at least two-fold increases ordecreases in the presence of the ETC inhibitor, 10 mM antimycin Acompared with the control lacking antimycin A. were identified, comparedto the Ceres full length cDNA and genomic sequence databanks, andequivalent Ceres clones identified. The MA_diff table reports theresults of this analysis, indicating those Ceres clones which are up ordown regulated over controls, thereby indicating the Ceres clones thatrepresent respiration responsive genes.

Examples of genes and gene products that are responsive to antimycin Ablock of respiration are shown in the Reference, Sequence, ProteinGroup, Protein Group Matrix, MA_diff and MA_clust tables. Whilerespiration responsive polynucleotides and gene products can act alone,combinations of these polynucleotides also affect growth anddevelopment. Useful combinations include different respirationresponsive polynucleotides and/or gene products that have similartranscription profiles or similar biological activities, and members ofthe same or similar biochemical pathways. Here, in addition topolynucleotides having similar transcription profiles and/or biologicalactivities, useful combinations include polynucleotides that may havedifferent transcription profiles but which participate in common oroverlapping pathways. Whole pathways or segments of pathways arecontrolled by transcription factor proteins and proteins controlling theactivity of signal transduction pathways. Therefore, manipulation ofsuch protein levels is especially useful for altering phenotypes andbiochemical activities of plants. Manipulation of one or morerespiration responsive gene activities are useful to modulate thebiological processes and/or phenotypes listed below.

Such respiration responsive genes and gene products can function toeither increase or dampen the phenotypes or activities below. Further,promoters of respiration responsive genes, as described in the Referencetables, for example, are useful to modulate transcription that isinduced by respiration or any of the following phenotypes or biologicalactivities below. Further, any desired sequence can be transcribed insimilar temporal, tissue, or environmentally specific patterns as therespiration responsive genes when the desired sequence is operablylinked to a promoter of a respiration responsive gene. The MA_diffTable(s) reports the transcript levels of the experiment (see EXPT ID:Mitchondria-Electron Transport (relating to SMD 8061, SMD 8063)). Fortranscripts that had higher levels in the samples than the control, a“+” is shown. A “−” is shown for when transcript levels were reduced inroot tips as compared to the control. For more experimental detail seethe Example section below.

Mitchondria-Electron Transport genes are those sequences that showeddifferential expression as compared to controls, namely those sequencesidentified in the MA_diff tables with a “+” or “−” indication.

Mitchondria-Electron Transport Genes Identified by Cluster Analyses ofDifferential Expression

Mitchondria-Electron Transport Genes Identified by Correlation to Genesthat are Differentially Expressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of Mitchondria-Electron Transport genes is anygroup in the MA_clust that comprises a cDNA ID that also appears in ExptID Mitchondria-Electron Transport (relating to SMD 8061, SMD 8063) ofthe MA_diff table(s).

Mitchondria-Electron Transport Genes Identified by Correlation to Genesthat Cause Physiological Consequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks ofMitchondria-Electron Transport genes. A group in the MA_clust isconsidered a Mitchondria-Electron Transport pathway or network if thegroup comprises a cDNA ID that also appears in Knock-in or Knock-outtables that causes one or more of the phenotypes described in sectionabove.

Mitchondria-Electron Transport Genes Identified By Amino Acid SequenceSimilarity

Mitchondria-Electron Transport genes from other plant species typicallyencode polypeptides that share amino acid similarity to the sequencesencoded by corn and Arabidopsis Mitchondria-Electron Transport genes.Groups of Mitchondria-Electron Transport genes are identified in theProtein Group table. In this table, any protein group that comprises apeptide ID that corresponds to a cDNA ID member of aMitchondria-Electron Transport pathway or network is a group of proteinsthat also exhibits Mitchondria-Electron Transport functions/utilities.

III.D.5.a. Use of Respiration Responsive Genes to Modulate Phenotypes

Respiration responsive genes and gene products are useful to or modulateone or more phenotypes including catabolism; energy generation, ATP,etc.; growth rate; whole plant, including height, flowering time, etc.;organs; flowers; fruits; stems; leaves; roots, lateral roots; biomass;fresh and dry weight during any time in plant life, such as maturation;number, size, and weight of flowers; seeds; branches; leaves; totalplant nitrogen content; amino acid/protein content of whole plant orparts; seed yield (such as number, size weight, harvest index, andcontent and composition, e.g., amino acid, nitrogen, oil, protein, andcarbohydrate); fruit yield; number, size, weight, harvest index; contentand composition, e.g., amino acid, nitrogen, oil, protein, carbohydrate,water; and photosynthesis (such as carbon dioxide fixation).

To improve any of the phenotype(s) above, activities of one or more ofthe respiration responsive genes or gene products can be modulated andthe plants tested by screening for the desired trait. Specifically, thegene, mRNA levels, or protein levels can be altered in a plant utilizingthe procedures described herein and the phenotypes can be assayed. As anexample, a plant can be transformed according to Bechtold and Pelletier(1998, Methods. Mol. Biol. 82:259-266) and/or screened for variants asin Winkler et al. (1998) Plant Physiol 118: 743-50 and visuallyinspected for the desired phenotype or metabolically and/or functionallyassayed according to Saito et al. (1994, Plant Physiol. 106: 887-95),Takahashi et al (1997, Proc. Natl. Acad. Sci. USA 94: 11102-07) andKoprivova et al. (2000, Plant Physiol. 122: 737-46).

III.D.5.b. Use of Respiration-Responsive Genes to Modulate BiochemicalActivities

The activities of one or more of the respiration responsive genes can bemodulated to change biochemical or metabolic activities and/or pathwayssuch as those noted below. Such biological activities can be measuredaccording to the citations included in the Table below:

BIOCHEMICAL OR METABOLIC ACTIVITIES CITATIONS PROCESS AND/OR PATHWAYSINCLUDING ASSAYS Respiration and Mitochondrial Electron Passam et al.(1973) energy-related Transport Chain Biochem Biophys. Acta processes325: 54-61 Alternative oxidase pathway Saisho et al. (1997) Plant Mol.Biol. 35: 585-600 Vanlerberghe and McIntosh (1994) Plant Physiol. 105:867-874 ATP generation pathways Mahler and Cordes (1966) ATP utilizationpathways In Biological Chemistry, Harper and Row Chloroplast energyrelated Foyer et al. (1989) Arch. pathways Biochem. Biophys. 268:687-697 Mills et al. (1978) Biochem. Biophys. Acta 504: 298-309Peroxisome energy related Olsen (1998) Plant mol. pathways Biol. 38:163-89 Cytoplasmic energy related Roberts et al. (1995) Febs Letterspathways 373: 307-309 Catabolism and Anabolism Mahler and Cordes (1966)In Biological Chemistry, Harper and Row Aerobic versus anaerobic Mahlerand Cordes (1966) In pathways Biological Chemistry, Harper and RowCoordination of Light-regulation of major Lam et al. (1998) Plant J.16(3): Carbon and Nitrogen central carbon and nitrogen 345-353Metabolism Metabolic pathways to Lejay et al. (1999) Plant J. 18(5):coordinate growth 509-519; and Oliveira et al. (1999) Plant. Phys. 121:301-309 Carbohydrate and nitrogen Lam et al. (1998) Plant J. 16(3):control of carbohydrate and 345-353 organic nitrogen accumulation Lejayet al. (1999) Plant J. 18(5): pathways 509-519; and Oliveira et al.(1999) Plant. Phys. 121: 301-309

Other biological activities that can be modulated by the respirationgenes and gene products are listed in the REF Tables. Assays fordetecting such biological activities are described in the Protein Domaintable.

Respiration responsive genes are differentially expressed in response toinhibition of mitochondrial electron transport by antimycin A. TheMA_diff table reports the changes in transcript levels of variousrespiration responsive genes that are differentially expressed inresponse to this treatment.

Profiles of these different respiration genes are shown in the Tablebelow with examples of associated biological activities.

EXAMPLES TRANSCRIPT PHYSIOLOGICAL OF BIOCHEMICAL LEVELS TYPE OF GENESCONSEQUENCES ACTIVITY Up regulated Responders to Changes in Transporterstranscripts inhibition of generation of ATP Catabolic and mitochondrialAlternate oxidase anabolic enzymes electron transport induction Changesin cell respiration Changes in and organelle Genes induced by catabolicand membrane inhibition of anabolic enzymes structures and mitochondrialand pathways potentials electron transport Specific gene Kinases andtranscription phosphatases initiation Transcription Changes in electronactivators transport proteins Change in chromatin structure and/orlocalized DNA topology Redox control Down-regulated Responders toChanges in ATP Transcription transcripts inhibition of generatingfactors mitochondrial pathways Change in protein electron transportChanges in structure by Genes repressed by pathways and phosphorylationinhibition of processes operating in (kinases) or mitochondrial cellsdephosphoryaltion electron transport Induction of (phosphatases) aerobicpathways Transporters Changes in Catabolic and catabolism and anabolicenzymes anabolism Changes in cell and organelle membrane structures andpotentials Change in chromatin structure and/or localized DNA topologychanges Stability factors for protein synthesis and degradationMetabolic enzymes Changes in redox Changes in redox activities enzymes

Use of Promoters of Respiration Genes

Promoters of Respiration genes are useful for transcription of anydesired polynucleotide or plant or non-plant origin. Further, anydesired sequence can be transcribed in a similar temporal, tissue, orenvironmentally specific patterns as the Respiration genes where thedesired sequence is operably linked to a promoter of a Respiration gene.The protein product of such a polynucleotide is usually synthesized inthe same cells, in response to the same stimuli as the protein productof the gene from which the promoter was derived. Such promoter are alsouseful to produce antisense mRNAs to down-regulate the product ofproteins, or to produce sense mRNAs to down-regulate mRNAs via sensesuppression.

III.D.6. Protein Degradation Genes, Gene Components and Products

One of the components of molecular mechanisms that operate to supportplant development is the “removal” of a gene product from a particulardevelopmental circuit once the substrate protein is not functionallyrelevant anymore in temporal and/or spatial contexts. The “removal”mechanisms can be accomplished either by protein inactivation (e.g.,phosphorylation or protein-protein interaction) or protein degradationmost notably via ubiquitination-proteasome pathway. Theubiquitination-proteasome pathway is responsible for the degradation ofa plethora of proteins involved in cell cycle, cell division,transcription, and signal transduction, all of which are required fornormal cellular functions. Ubiquitination occurs through the activity ofubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2),and ubiquitin-protein ligases (E3), which act sequentially to catalyzethe attachment of ubiquitin (or other modifying molecules that arerelated to ubiquitin) to substrate proteins (Hochstrasser 2000, Science289: 563). Ubiquitinated proteins are then routed to proteasomes fordegradation processing [2000, Biochemistry and Molecular Biology ofPlants, Buchanan, Gruissem, and Russel (eds), Amer. Soc. of PlantPhysiologists, Rockville, Md.]. The degradation mechanism can beselective and specific to the concerned target protein (Joazeiro andHunter 2001, Science 289: 2061; Sakamoto et al., 2001, PNAS Online141230798). This selectivity and specificity may be one of the ways thatthe activity of gene products is modulated.

III.D.6.a. Identification of Protein Degradation Genes Gene Componentsand Products

“Protein degradation” genes identified herein are defined as genes, genecomponents and products associated with or dependant on theubiquitination—proteasome protein degradation process. Examples of such“protein degradation” genes and gene products are shown in the Referenceand Sequence Tables. The biochemical functions of the protein productsof many of these genes are also given in the Reference, Sequence,Protein Group, Protein Group Matrix tables, MA_diff and MA_clust tables.Selected genes, gene components and gene products of the invention canbe used to modulate many plant traits from architecture to yield tostress tolerance.

“Protein Degradation” Genes, Gene Components and Products Identified byPhenotypic Observations

“Protein degradation” genes herein were discovered and characterizedfrom a much larger set of genes in experiments designed to find thegenes associated with the increased number of lateral branches (andsecondary inflorescences) that are formed per cauline node. In theseexperiments, “protein degradation” genes were identified using a mutantwith these characteristics. The gene causing the changes was identifiedfrom the mutant gene carrying an inserted tag. The mutant plant wasnamed 13B12-1 and the mutant was in the E2 conjugating enzyme gene ofthe ubiquitination process. Compared to “wild-type” parental plants, themutant plants exhibited multiple lateral stems per node andmulti-pistillated flowers. For more experimental detail, see Examplesection below.

Protein Degradation Genes, Gene Components and Products Identified byDifferential Expression

“Protein degradation” genes were also identified by measuring therelative levels of mRNA products in the mutant plant 13B12-1 lacking theE2 conjugating enzyme versus a “wild-type” parental plant. Specifically,mRNAs were isolated from 13B12-1 and compared with mRNAs isolated fromwild-type plants utilizing microarray procedures. The MA_diff Table(s)reports the transcript levels of the experiment (see EXPT ID: 108451).For transcripts that had higher levels in the samples than the control,a “+” is shown. A “−” is shown for when transcript levels were reducedin root tips as compared to the control. For more experimental detailsee the Example section below.

Protein Degradation genes are those sequences that showed differentialexpression as compared to controls, namely those sequences identified inthe MA_diff tables with a “+” or “−” indication.

Protein Degradation Genes Identified by Cluster Analyses of DifferentialExpression

Protein Degradation Genes Identified by Correlation to Genes that areDifferentially Expressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of Protein Degradation genes is any group in theMA_clust that comprises a cDNA ID that also appears in Expt ID 108451 ofthe MA_diff table(s).

Protein Degradation Genes Identified by Correlation to Genes that CausePhysiological Consequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of ProteinDegradation genes. A group in the MA_clust is considered a ProteinDegradation pathway or network if the group comprises a cDNA ID thatalso appears in Knock-in or Knock-out tables that causes one or more ofthe phenotypes described in section above.

Protein Degradation Genes Identified By Amino Acid Sequence Similarity

Protein Degradation genes from other plant species typically encodepolypeptides that share amino acid similarity to the sequences encodedby corn and Arabidopsis Protein Degradation genes. Groups of ProteinDegradation genes are identified in the Protein Group table. In thistable, any protein group that comprises a peptide ID that corresponds toa cDNA ID member of a Protein Degradation pathway or network is a groupof proteins that also exhibits Protein Degradation functions/utilities.

These differentially expressed genes include genes associated with thedegradation process and the genes whose expression is disturbed by theaberrant ubiquitination.

Examples of phenotypes, biochemical activities, and transcriptionprofiles that can be modulated using these genes, gene components andgene products are described above and below.

III.D.6.b. Use of “Protein Degradation” Genes, Gene Components andProducts to Modulate Phenotypes

The “protein degradation” genes, their components and products of theinstant invention are useful for modulating one or more processesrequired for post-translational modification (e.g., ubiquitination) anddegradation or inactivation of substrate proteins and also the pathwaysand processes that are associated with protein inactivation that areimportant for either or all of the following: (i) cell proliferation;(ii) cell differentiation; and (iii) cell death. The “proteindegradation” genes, gene components and gene products are useful toalter or modulate one or more phenotypes including cell proliferationand cell size.

The intracellular levels of many proteins are regulated byubiquitin-proteasome proteolysis. Without proper regulation of proteinlevels, normal cell differentiation can be altered. Examples of celldifferentiation and development can be modulated by the genes and geneproducts of this invention include root size (such as length of primaryroots or length of lateral roots) and function; branching and stemformation (such as multiple pistils, multiple lateral stems or secondaryinflorescence per cauline node, and internode length) and celldifferentiation and/or development in response to hormones (such asAuxin).

Programmed cell death can result from specific and targeted degradationof critical substrate proteins (e.g., transcription factors, enzymes,and proteins involved in signal transduction). Thus, alteration of“protein degradation” genes, their gene products, and the correspondingsubstrate proteins that they are acting upon are useful to modulate thevigor and yield of the plant overall as well as distinct cells, organs,or tissues. Traits that can be modulated by these genes and geneproducts include sterility or reproduction and seedling lethality.

Uses of Plants that are Modified as Described Above

Genes that control fundamental steps in regulatory pathways, such asprotein inactivation, that in turn influence cascades and networks ofother genes and processes are extremely useful. They and their componentparts can be used selectively to manipulate development in specificcells, tissues and organs, including meristems when genes are designedto inactivate the normal genes only in specific cells, tissues andorgans or to promote protein production where it is not normallyproduced. They can also be used to promote/control cell death.

Other “protein degradation” genes described here are components of thepathways determining organ identity and phenotypes. These and theircomponent parts are also useful for modifying the characteristics ofspecific cells, tissues and organs when regulated appropriately. Thus“protein degradation” genes have wide utility for achieving thefollowing: better plant survival by decreased lodging; better responsesto high plant density; better stress tolerance; better animal (includinghuman) nutrition values; improved dietary mineral nutrition; more vigor,growth rate and yield in terms of biomass; root/tuber yield (in terms ofnumber, size, weight, or harvest index); content and composition, e.g.amino acid, jasmonate, oil, protein and starch; number of flowers; seedyield (e.g. number, size, weight, harvest index, content andcomposition, e.g. amino acid, jasmonate, oil, protein and starch); andfruit yield (e.g. number, size, weight, harvest index, post harvestquality, content and composition, e.g. amino acid, jasmonate, oil,protein and starch).

To regulate any of the phenotype(s) above, activities of one or more ofthe “protein degradation” genes or gene products can be modulated andtested by screening for the desired trait. Specifically, the gene, mRNAlevels, or protein levels can be altered in a plant utilizing theprocedures described herein and the phenotypes can be assayed. Inaddition, a synthetic molecule containing specific domains from “proteindegradation” genes or gene product and/or in combination with otherdomains from gene products that are not necessarily related to proteindegradation pathway can be constructed to target the degradation orinactivation of specific substrate proteins. As an example, a plant canbe transformed according to Bechtold and Pelletier (1998, Methods. Mol.Biol. 82:259-266) and/or screened for variants as in Winkler et al.(1998) Plant Physiol 118: 743-50 and visually inspected for the desiredphenotype or metabolically and/or functionally assayed according toDolan et al. (1993, Development 119: 71-84), Dolan et al. (1997,Development 124: 1789-98), Crawford and Glass (1998, Trends PlantScience 3: 389-95), Wang et al. (1998, PNAS USA 95: 15134-39), Gaxiolaet al. (1998, PNAS USA 95: 4046-50), Apse et al. (1999, Science 285:1256-58), Fisher and Long (1992, Nature 357: 655-60), Schneider et al.(1998, Genes Devel 12: 2013-21) and Hirsch (1999, Curr Opin Plant Biol.2: 320-326).

Use of Protein Degradation Genes, Gene Components and Products toModulate Biochemical Activities

One or more of the “protein degradation” genes and their components canbe used to modulate biochemical or metabolic activities, processesand/or pathways such as those noted below. Such biological activitiescan be measured according to the citations included in the Table below:

BIOCHEMICAL OR METABOLIC ACTIVITIES CITATIONS INCLUDING PROCESS AND/ORPATHWAYS ASSAYS Growth, Differentiation and Auxin response Schwechheimeret al, Science 292: Development 1379 (2001); Leyser et al, Nature 8: 161(1993); Lasswell et al, Plant Cell 12: 2395 (2000) Photomorphogenesisvia leaf Schwechheimer et al, Science 292: cells and meristems 1379(2001) Apical dominance via shoot Schwechheimer et al, Science 292:meristems 1379 (2001) Lateral root development via root Xie et al, GenesDev 14: 3024 meristem (2000) Hypocotyl, shoot elongation by Nagpal etal, Plant Physiol 123: 563 hormone controlled process (2000) GeneExpression and related mRNA stability Johnson et al, PNAS 97: 13991cellular processes (2000); Gene activation Pham and Sauer, 289: 2357(2000) Cell division and cell cycle King et al, Cell 81: 279 (1995);control in meristems Ciechanover et al, Cell 37: 57 (1984); Finley etal, Cell 37: 43 (1984); Robzyk et al, Science 287: 501 (2000) Chromatinremodeling Roest et al, Cell 86: 799 (1996) Post-translationalmodification Biederer et al, Science 278: 1806 and organelle targetingof (1997) proteins

Other biological activities that can be modulated by the “proteindegradation” gene, gene components and products are listed in theReference tables. Assays for detecting such biological activities aredescribed in the Protein Domain table.

III.D.6.d. Use of Protein Degradation Genes, Gene Components andProducts to Modulate Transcription Levels of Other Genes

The expression of many genes is “up regulated” or “down regulated” inthe 13B12-1 mutant because some protein degradation genes and theirproducts are integrated into complex networks that regulatetranscription of many other genes. Some protein degradation genes aretherefore useful for modifying the transcription of other genes andhence complex phenotypes, as described above. Profiles of “proteindegradation” genes are described in the Table below with associatedbiological activities. “Up-regulated” profiles are those where the geneproduces mRNA levels that are higher in the 13B12-1 as compared towild-type plant; and vice-versa for “down-regulated” profiles.

EXAMPLES OF TYPE OF GENES PHYSIOLOGICAL BIOCHEMICAL WHOSE CONSEQUENCESOF ACTIVITIES WHOSE TRANSCRIPT TRANSCRIPTS MODIFYING GENE TRANSCRIPTSARE LEVELS ARE CHANGED PRODUCT LEVELS CHANGED Up Regulated Genes inducedas Shoot formation Transcription Transcripts a consequence of Lateralstem, lateral Activators and mutant and main Repressors ubiquitinationinflorescence Chromatin Structure degradation development and/orLocalized system Internode DNA Topology Genes repressed elongationdetermining proteins by “protein Node determination Methylated DNAdegradation” and development binding proteins system directly or Rootformation Kinases, indirectly Lateral root Phosphatases Genes represseddevelopment Signal transduction or mRNAs Proper response to pathwayproteins degraded as a Auxin and other Transporters consequence ofgrowth regulators Metabolic Enzymes mutant Seed dormancy and Cell cycleubiquitination seed development checkpoint proteins degradationResistance to Cell Membrane process drought and other Structure Andforms of stress Proteins Secondary Cell Wall Proteins metaboliteProteins involved in biosynthesis secondary metabolism Seed storagemetabolism Down Regulated Genes activated Transcripts by “proteindegradation” systems directly or indirectly

“Protein degradation” genes and gene products can be modulated alone orin combination as described in the introduction. Of particular interestare combination of these genes and gene products with those thatmodulate hormone responses and/or metabolism. Hormone responsive andmetabolism genes and gene products are described in more detail in thesections above. Such modification can lead to major changes in plantarchitecture and yield.

Use of Promoters and “Protein Degradation Genes, Gene Components andProducts”

Promoters of “protein degradation” genes, as described in the Referencetables, for example, can be used to modulate transcription of anypolynucleotide, plant or non plant to achieve synthesis of a protein inassociation with production of the ubiquitination-proteasome pathway orthe various cellular systems associated with it. Additionally suchpromoters can be used to synthesize antisense RNA copies of any gene toreduce the amount of protein product produced, or to synthesize RNAcopies that reduce protein formation by RNA interference. Suchmodifications can make phenotypic changes and produce altered plants asdescribed above.

III.D.7. Carotenogenesis Responsive Genes, Gene Components and Products

Carotenoids serve important biochemical functions in both plants andanimals. In plants, carotenoids function as accessory light harvestingpigments for photosynthesis and to protect chloroplasts and photosystemII from heat and oxidative damage by dissipating energy and scavengingoxygen radicals produced by high light intensities and other oxidativestresses. Decreases in yield frequently occur as a result of lightstress and oxidative stress in the normal growth ranges of crop species.In addition light stress limits the geographic range of many cropspecies. Modest increases in oxidative stress tolerance would greatlyimprove the performance and growth range of many crop species. Thedevelopment of genotypes with increased tolerance to light and oxidativestress would provide a more reliable means to minimize crop losses anddiminish the use of energy-costly practices to modify the soilenvironment.

In animals carotenoids such as beta-carotene are essential provitaminsrequired for proper visual development and function. In addition, theirantioxidative properties are also thought to provide valuable protectionfrom diseases such as cancer. Modest increases in carotenoid levels incrop species could produce a dramatic effect on plant nutritionalquality. The development of genotypes with increased carotenoid contentwould provide a more reliable and effective nutritional source ofVitamin A and other carotenoid derived antioxidants than through the useof costly nutritional supplements.

Genetic changes produced through DNA mutation in a plant can result inthe modulation of many genes and gene products. Examples of suchmutation altered genes and gene products are shown in the Reference andSequence Tables. These genes and/or products are responsible for effectson traits such as plant vigor, nutritional content and seed yield.

While carotenoid synthesis and/or oxidative stress responsivepolynucleotides and gene products can act alone, combinations of thesepolynucleotides also affect growth and development. Useful combinationsinclude different carotenoid biosynthetic polynucleotides and/or geneproducts that have similar transcription profiles or similar biologicalactivities, and members of the same or similar biochemical pathways. Inaddition, the combination of an carotenoid synthesis or oxidative stressprotective polynucleotide and/or gene product with anotherenvironmentally responsive polynucleotide is also useful because of theinteractions that exist between hormone-regulated pathways, stresspathways, nutritional pathways and development. Here, in addition topolynucleotides having similar transcription profiles and/or biologicalactivities, useful combinations include polynucleotides that may havedifferent transcription profiles but which participate in a commonpathway.

Such carotenoid synthesis/oxidative stress tolerance genes and geneproducts can function to either increase or dampen the above phenotypesor activities either in response to changes in light intensity or in theabsence of osmotic fluctuations. They were discovered and characterizedfrom a much larger set of genes by experiments designed to find geneswhose mRNA products participate in carotenogenesis. These experimentsmade use of an Arabidopsis mutant (Or) having an accumulation of up to500 times more beta-carotene than wild-type in non-photosynthetictissues.

Microarray technology allows monitoring of gene expression levels forthousands of genes in a single experiment. This is achieved byhybridizing labeled fluorescent cDNA pools to glass slides that containspots of DNA (Schena et al. (1995) Science 270: 467-70). TheUSArabidopsis Functional Genomics Consortium (AFGC) has recently madepublic the results from such microarray experiments conducted with AFGCchips containing some 10,000 non-redundant ESTs, selected from about37,000 randomly sequenced ESTs generated from mRNA of different tissuesand developmental stages.

The sequences of the ESTs showing at least two-fold increases ordecreases in the mutant plant compared with wild type seedlings wereidentified, compared to the Ceres full length cDNA and genomic sequencedatabanks, and equivalent Ceres clones identified. MA_diff Table reportsthe results of this analysis, indicating those Ceres clones which are upor down regulated over controls, thereby indicating the Ceres cloneswhich represent Carotenoid synthesis/oxidative stress toleranceresponsive genes. The MA_diff Table(s) reports the transcript levels ofthe experiment (see EXPT ID: Cauliflower (relating to SMD 5329, SMD5330)). For transcripts that had higher levels in the samples than thecontrol, a “+” is shown. A “−” is shown for when transcript levels werereduced in root tips as compared to the control. For more experimentaldetail see the Example section below.

Carotenogenesis genes are those sequences that showed differentialexpression as compared to controls, namely those sequences identified inthe MA diff tables with a “+” or “−” indication.

Carotenogenesis Genes Identified by Cluster Analyses of DifferentialExpression

Carotenogenesis Genes Identified by Correlation to Genes that areDifferentially Expressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of Carotenogenesis genes is any group in theMA_clust that comprises a cDNA ID that also appears in Expt IDCauliflower (relating to SMD 5329, SMD 5330) of the MA_diff table(s).

Carotenogenesis Genes Identified by Correlation to Genes that CausePhysiological Consequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks ofCarotenogenesis genes. A group in the MA_clust is considered aCarotenogenesis pathway or network if the group comprises a cDNA ID thatalso appears in Knock-in or Knock-out tables that causes one or more ofthe phenotypes described in section above.

Carotenogenesis Genes Identified by Amino Acid Sequence Similarity

Carotenogenesis genes from other plant species typically encodepolypeptides that share amino acid similarity to the sequences encodedby corn and Arabidopsis Carotenogenesis genes. Groups of Carotenogenesisgenes are identified in the Protein Group table. In this table, anyprotein group that comprises a peptide ID that corresponds to a cDNA IDmember of a Carotenogenesis pathway or network is a group of proteinsthat also exhibits Carotenogenesis functions/utilities.

III.D.7.a. Use of Carotenoid Synthesis/Oxidative Stress ToleranceResponsive Genes, Gene Components and Products to Modulate Phenotypes

Carotenoid synthesis/oxidative, stress tolerance genes and gene productsare useful to or modulate one or more phenotypes including growth rate;whole plant, including height, flowering time, etc.); seedling; organ(such as stem, leaves, roots, flowers, fruits, or seed yield, size, orweight); seed development; embryo; germination; cell differentiation;chloroplasts; plant nutrition; uptake and assimilation of organiccompounds; uptake and assimilation of inorganic compounds; animal(including human) nutrition; improved dietary mineral nutrition; stressresponses; drought; cold; and osmotic.

To improve any of the phenotype(s) above, activities of one or more ofthe Carotenoid synthesis/oxidative stress tolerance genes or geneproducts can be modulated and tested by screening for the desired trait.Specifically, the gene, mRNA levels, or protein levels can be altered ina plant utilizing the procedures described herein and the phenotypes canbe assayed. As an example, a plant can be transformed according toBechtold and Pelletier (1998, Methods. Mol. Biol. 82:259-266) and/orscreened for variants as in Winkler et al. (1998) Plant Physiol 118:743-50 and visually inspected for the desired phenotype or metabolicallyand/or functionally assayed according to Friedrich, (1999, JAMA 282:1508), Kumar et al. (1999, Phytochemistry 51: 847-51), La Rocca et al.(2000, Physiologia Plantarum 109: 51-7) and Bartley (1994, In: Ann RevPlant Physiol Plant Molec Biol, Jones and Somerville, eds, AnnualReviews Inc, Palo Alto, Calif.).

III.D.7.b. Use of Carotenoid Synthesis/Oxidative Stress ToleranceResponsive Genes, Gene Components and Products to Modulate BiochemicalActivities

The activities of one or more of the carotenoid synthesis/oxidativestress tolerance genes can be modulated to change biochemical ormetabolic activities and/or pathways such as those noted below. Suchbiological activities can be measured according to the citationsincluded in the Table below:

BIOCHEMICAL OR METABOLIC ACTIVITIES CITATIONS INCLUDING PROCESS AND/ORPATHWAYS ASSAYS Growth, Chloroplast biosynthesis Kumar et al. (1999)Phytochemistry Differentiation 51: 847-51 and Development Fraser et al.(1994) Plant Physiol 105: 405-13 Metabolism Carotenoid biosynthesisKumar et al. (1999) Phytochemistry 51: 847-51 Herbicide resistance LaRocca et al. (2000) Physiolgia Plantarum 109: 51-57 Regulate abscisicacid levels Tan et al. (1997) PNAS USA 94: 12235-40 Drought, cold andosmotic Tan et al. (1997) PNAS USA 94: tolerance 12235-40

Other biological activities that can be modulated by the Carotenoidsynthesis, oxidative stress tolerance genes and gene products are listedin the Reference Tables. Assays for detecting such biological activitiesare described in the Protein Domain table.

Profiles of these different carotenoid synthesis/oxidative stresstolerance responsive genes are shown in the Table below together withexamples of the kinds of associated biological activities.

EXAMPLES OF TRANSCRIPT PHYSIOLOGICAL BIOCHEMICAL LEVELS TYPE OF GENESCONSEQUENCES ACTIVITY Up regulated Genes induced during GeneTransporters transcripts carotenoid synthesis/ Repression/InductionMetabolic oxidative stress activity enzymes tolerance activity Cellcycle progression Kinases and Chromatin phosphatases condensationTranscription Synthesis of activators metabolites and/or Change inproteins chromatin Modulation of structure and/or transduction pathwayslocalized DNA Specific gene topology transcription initiationDown-regulated Genes repressed Gene Transcription transcripts duringcarotenoid repression/induction factors synthesis/oxidative activityChange in stress tolerance Changes in pathways protein structureactivity and processes by Genes with operating in cells phosphorylationdiscontinued Changes in (kinases) or expression or metabolism other thandephosphorylation unsTable mRNA in carotenoid (phosphatases) conditionsof reduced synthesis/oxidative Change in carotenoid stress tolerancechromatin synthesis/oxidative structure and/or stress tolerance DNAtopology Stability of factors for protein synthesis and degradationMetabolic enzymes

Use of Promoters of Carotenogenesis Responsive Genes

Promoters of Carotenogenesis responsive genes are useful fortranscription of any desired polynucleotide or plant or non-plantorigin. Further, any desired sequence can be transcribed in a similartemporal, tissue, or environmentally specific patterns as theCarotenogenesis responsive genes where the desired sequence is operablylinked to a promoter of a Carotenogenesis responsive gene. The proteinproduct of such a polynucleotide is usually synthesized in the samecells, in response to the same stimuli as the protein product of thegene from which the promoter was derived. Such promoter are also usefulto produce antisense mRNAs to down-regulate the product of proteins, orto produce sense mRNAs to down-regulate mRNAs via sense suppression.

III.D.8. Viability Genes, Gene Components and Products

Plants contain many proteins and pathways that when blocked or inducedlead to cell, organ or whole plant death. Gene variants that influencethese pathways can have profound effects on plant survival, vigor andperformance. The critical pathways include those concerned withmetabolism and development or protection against stresses, diseases andpests. They also include those involved in apoptosis and necrosis. Theapplicants have elucidated many such genes and pathways by discoveringgenes that when inactivated lead to cell or plant death.

Herbicides are, by definition, chemicals that cause death of tissues,organs and whole plants. The genes and pathways that are activated orinactivated by herbicides include those that cause cell death as well asthose that function to provide protection. The applicants haveelucidated these genes.

The genes defined in this section have many uses including manipulatingwhich cells, tissues and organs are selectively killed, which areprotected, making plants resistant to herbicides, discovering newherbicides and making plants resistant to various stresses.

III.D.8.a. Identification of Viability Genes, Gene Components andProducts

Viability genes identified here are defined as genes, gene componentsand products capable of inhibiting cell, tissue, organ or whole plantdeath or protecting cells, organs and plants against death and toxicchemicals or stresses. Examples of such viability genes and geneproducts are shown in the Reference, Sequence, Protein Group, ProteinGroup Matrix tables, MA_diff, MA_clust, Knock-in and Knock-out tables.The biochemical functions of the protein products of many of these genesdetermined from comparisons with known proteins are also given in theReference tables.

Viability Genes, Gene Components and Products Identified by PhenotypicObservations

These genes were discovered and characterized from a much larger set ofgenes by experiments designed to find genes that cause seriousdisturbances in progeny survival, seed germination, development, embryoand/or seedling growth. In these experiments, viability genes wereidentified by either (1) ectopic expression of a cDNA in a plant or (2)mutagenesis of a plant genome. The plants were then cultivated and oneor more of the following phenotypes, which varied from the parentalwild-type was observed:

-   -   A. Gametophytic loss of progeny seedlings (detected from a        parent on the basis of a linked herbicide resistance gene        showing abnormal segregation ratios, as revealed by treating        with herbicide)    -   B. Embryo death, resulting in some cases to loss of seed    -   C. Pigment variation in cotyledons and leaves, including absence        of chlorophyll, which leads to seedling death.        -   1. Abinos        -   2. Yellow/greens    -   D. Cotyledons produced but no or few leaves and followed by        seedling death.    -   E. Very small plantlets

The genes identified in these experiments are shown in Tables X.

Viability Genes Gene Components and Products Identified by DifferentialExpression

Viability genes were also identified from a much larger set of genes byexperiments designed to find genes whose mRNA products changed inconcentration in response to applications of different herbicides toplants. Viability genes are characteristically differentiallytranscribed in response to fluctuating herbicide levels orconcentrations, whether internal or external to an organism or cell. TheMA_diff Table reports the changes in transcript levels of variousviability genes in entire seedlings at 0, 4, 8, 12, 24, and 48 hoursafter a plant was sprayed with a Hoagland's nutrient solution enrichedwith either 2,4 D (Trimec), Glean, Grassgetter, Roundup, or Finaleherbicides as compared to seedlings sprayed with Hoagland's solutiononly.

The MA_diff Table(s) reports the transcript levels of the experiment(see EXPT ID: 108467, 107871, 107876, 108468, 107881, 108465, 107896,108466, 107886, 107891, 108501). For transcripts that had higher levelsin the samples than the control, a “+” is shown. A “−” is shown for whentranscript levels were reduced in root tips as compared to the control.For more experimental detail see the Example section below.

Viability genes are those sequences that showed differential expressionas compared to controls, namely those sequences identified in theMA_diff tables with a “+” or “−” indication.

Viability Genes Identified by Cluster Analyses of DifferentialExpression

Viability Genes Identified by Correlation to Genes that areDifferentially Expressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of Viability genes is any group in the MA_clustthat comprises a cDNA ID that also appears in Expt ID 108467, 107871,107876, 108468, 107881, 108465, 107896, 108466, 107886, 107891, 108501of the MA_diff table(s).

Viability Genes Identified by Correlation to Genes that CausePhysiological Consequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of Viabilitygenes. A group in the MA_clust is considered a Viability pathway ornetwork if the group comprises a cDNA ID that also appears in Knock-inor Knock-out tables that causes one or more of the phenotypes describedin section above.

Viability Genes Identified by Amino Acid Sequence Similarity

Viability genes from other plant species typically encode polypeptidesthat share amino acid similarity to the sequences encoded by corn andArabidopsis Viability genes. Groups of Viability genes are identified inthe Protein Group table. In this table, any protein group that comprisesa peptide ID that corresponds to a cDNA ID member of a Viability pathwayor network is a group of proteins that also exhibits Viabilityfunctions/utilities.

It is assumed that those gene activity changes in response to the toxicherbicides are either responsible, directly or indirectly, for celldeath or reflect activation of defense pathways. These genes aretherefore useful for controlling plant viability.

Examples of phenotypes, biochemical activities, or transcript profilesthat can be modulated using selected viability gene components aredescribed above and below.

III.D.8.b. Use of Viability Genes, Gene Components and Products toModulate Phenotypes

Deficiencies in viability genes can cause cell death at various ratesand under various conditions. Viability genes can be divided into twoclasses; (1) those that lead to cell death under permissive growthconditions and (2) those that cause cell demise under restrictiveconditions. Examples of the first class are viability genes which encodetoxins or which participate in the programmed cell death pathway(s).Disruption of metabolic pathways, such as amino acid synthesis, may notcause death when the cell is supplemented with appropriate amino acids,but can cause death under more restrictive conditions.

Some deficiencies in viability genes identified cause the organism as awhole to die, while other genes cause death only of a specific subset ofcells or organs. For example, genes identified from embryo viabilityphenotypes can cause an entire organism to die. In contrast, genescharacterized from gametophytic lethals may inhibit cell growth only ina select set of cells. In addition, some viability genes may not causean immediate demise. A seedling lethal phenotype is one such example,where a seed germinates and produces cotyledons but the plant diesbefore producing any true leaves. Yellow-green pigment mutants provideyet another set of examples. In some cases, the plant produces a numberof yellow-green leaves but dies before producing any seed, due in part,to the necessity to produce chlorophyll in functioning chloroplasts tofix CO₂.

Viability genes, in which mutational deficiencies lead to death, carryno duplicates in the haploid plant genome. They thus may be especiallylikely to promote viability and vigor when expressed more optimally in aplant, in specific tissues or throughout the plant.

Proteins which lead to death when inactivated, and other proteins in thepathways in which they act, are potential targets for herbicides. Inthis kind of application, chemicals specifically capable of interactingwith such proteins are discovered. Typically, this could be done bydesigning a gene involving the relevant viability gene, that alsofacilitates a rapid easily measured assay for the functioning of theprotein product, and treating plants containing the new genes with thepotential herbicides. Those chemicals specifically interfering with theprotein activity can then easily be selected for further development.

Genes whose products interact directly with a herbicide can also bemodified such that the herbicide no longer inactivates the protein. Suchgenes are useful for making herbicide resistant plants, valuable inagriculture.

Many of the genes activated or inactivated by the herbicides definegenes involved in the pathways that protect the plant against damage andstresses. These genes and gene components, especially those regulatingsuch pathways, are especially useful for enhancing the ability of plantsto withstand specific stresses, including herbicides. [See the sectionson Stress responsive genes, gene components and products.]

Genes that cause cellular death can be used to design new genes thatcause death of specific cells and tissues and hence new valuableproducts. For example, activation of genes causing death in cellsspecifying seeds can be used to produce fruits lacking seeds. They canalso be used to prevent cell death by pathogens and pests.

The genes and gene components of the instant invention are useful tomodulate one or more processes that affect viability and vigor at the(1) cellular level; (2) organelle level; (3) organ level; or (4) overallorganism level.

Phenotypes that are modulated by these genese and gene componentsinclude (1) at the cellular level: cell size, cell differentiation, celldivision, cell longevity, cell position, and cytotoxins; (2) at theorganelle level: chloroplasts and/or mitochondria; (3) at the organlevel: flower number or size; seed size, number or composition (aminoAcid, carbohydrates, lipid, and secondary metabolites); fruit size,number, or composition (amino Acid, carbohydrates, lipid, and secondarymetabolites); fruit drop, fruit ripening; leaf (size, composition, aminoacid, carbohydrates, lipid, and secondary metabolites, photoefficiency,abscission, or senescence); stem; or root; and (4) at the overallorganism level: vigor (e.g. increased biomass), stress tolerance (e.g.cold, drought, heat, herbicide, oxidative, and salt); and pathogenresistance

To regulate any of the phenotype(s) above, activities of one or more ofthe viability genes or gene products can be modulated in an organism andtested by screening for the desired trait. Specifically, the gene, mRNAlevels, or protein levels can be altered in a plant utilizing theprocedures described herein and the phenotypes can be assayed. As anexample, a plant can be transformed according to Bechtold and Pelletier(Methods. Mol. Biol. 82:259-266 (1998)) and/or screened for variants asin Winkler et al., Plant Physiol. 118: 743-50 and visually inspected forthe desired phenotype or metabolically and/or functionally assayed.

III.D.8.c. Use of Viability Genes, Gene Components and Products toModulate Biochemical Activities

The viability genes, their components and/or products can be used tomodulate processes, biochemical or metabolic activities and/or pathwayssuch as those noted below. Such biological activities can be measuredaccording to the citations included in the table below:

BIOCHEMICAL OR METABOLIC ACTIVITIES CITATIONS INCLUDING PROCESS AND/ORPATHWAYS ASSAYS Amino Acid Synthesis Aceto-lactate synthase Hershey etal. (1999) Plant Mol. Biol. 40, 795-806 Cell Wall Synthesis Cellulosesynthase Peng et al. (2001) Plant Physiol. 126, 981-982 Kawagoe andDelmer (1997) Genet Eng. 19, 63-87 Nucleotide Synthesis Coenzyme Abiosynthesis Kupke et al. (2001) J. Biol. Chem. 276, 19190-19196 LipidSynthesis Oleosin biosynthesis Singh et al. (2000) Biochem. Soc. Trans.28, 925-927 Zou et al. (1996). Plant Mol. Biol. 31, 429-433 HormoneSignaling Brassinolide and light signal Kang et al. (2001) Cell 105,Pathways transduction 625-636 Hormone Biosynthesis Cytokininbiosynthesis Takei et al, (2001) J. Biol. Chem. 276, 26405-26410Secondary Metabolites Carotenoid biosynthesis Estevez et al. (2001) J.Biol. Chem. 276, 22901-22909 Carol and Kuntz (2001) Trendy Plant Sci. 6,31-36 Pogson and Rissler (2001) Phil. Trans. Roy. Soc. Lord. B 355,1395-1400 Clearing of Toxic Ubiquitination Substances Growth,Differentiation Farnesylation Pei et al (1998) Science 282: AndDevelopment Nitrogen Metabolism 287-290; Cutler et al. (1996) Science273: 1239 Goupil et al (1998) J Exptl Botany 49: 1855-62 WaterConservation And Stomatal Development And Allen et al. (1999) PlantResistance To Drought Physiology Cell 11: 1785-1798 And Other RelatedStress Response Pathways Li et al. 2000 Science 287: Stresses InhibitionOf Ethylene 300-303 Production Under Low Water Burnett Et Al 2000. J.Exptl Potential Botany 51: 197-205 Proline And Other Osmolite Raschke(1987) In: Stomatal Synthesis And Degradation Function Zeiger et al.Eds., 253-279 Bush And Pages (1998) Plant Mol. Biol. 37: 425-35 SpollenEt Al (2000) Plant Physiol. 122: 967-976 Hare et al. (1998) Plant, CellAnd Environment 21: 535-553; Hare et al. (1999) J. Exptl. Botany 50:413-434 Programmed cell death Proteases Kamens et al. (1995) J. Biol.DNA endonucleases Chem. 270, 15250-15256 Mitochondriae uncoupling Wanget al. (2001) proteins Anticancer Res. 21, 1789-1794 Drake et al. (1996)Plant Mol. Biol 304, 755-767 Mittler and Lam (1995) Plant Cell 7,1951-1962 Mittler and Lam (1995) Plant Physiol. 108, 489-493 Thelen andNorthcote (1989) Planta 179, 181-195 Hanak and Jezek (2001) FEBS Lett.495, 137-141 Plasmalemma and Tonoplast Macrobbie (1998) Philos IonChannel Changes Trans R Soc Lond B Biol Ca2+ Accumulation Sci 353:1475-88; Li et al K+ Efflux (2000) Science 287: 300-303; Activation OfKinases And Barkla et al. (1999) Phosphatases Plant Physiol. 120:811-819 Lacombe et al. (2000) Plant Cell 12: 837-51; Wang et al. (1998)Plant Physiol 118: 1421-1429; Shi et al. (1999) Plant Cell 11: 2393-2406Gaymard et al. (1998) Cell 94: 647-655 Jonak et al. (1996) Proc. Natl.Acad. Sci 93: 11274-79; Sheen (1998) Proc. Natl. Acad. Sci. 95: 975-80;Allen et al. (1999) Plant Cell 11: 1785-98

Other biological activities that can be modulated by the viabilitygenes, their components and products are listed in the Reference tables.Assays for detecting such biological activities are described in theProtein Domain table.

III.D.8.d. Use of Viability Genes, Gene Components and Products toModulate Transcript Levels of Other Genes

The expression of many genes is “up regulated” or “down regulated”following herbicide treatment and also in the leaf mutants, because some“viability” genes and their products are integrated into complexnetworks that regulate transcription of many other genes. Some“viability genes” are therefore useful for modifying the transcriptionof other genes and hence complex phenotypes, as described above. Thedata from differential expression experiments can be used to identify anumber of types of transcript profiles of “viability genes”, including“early responders,” and “delayed responders”, “early responderrepressors” and “delayed repressors”. Profiles of these different typesresponsive genes are shown in the Table below together with examples ofthe kinds of associated biological activities. “Up-regulated” profilesare those where the mRNA transcript levels are higher in the herbicidetreated plants as compared to the untreated plants. “Down-regulated”profiles represent higher transcript levels in the untreated plant ascompared to the herbicide treated plants.

PHYSIOLOGICAL EXAMPLES OF TYPE OF GENES CONSEQUENCES BIOCHEMICAL WHOSEOF MODIFYING ACTIVITIES WHOSE TRANSCRIPT TRANSCRIPTS ARE GENE PRODUCTTRANSCRIPTS ARE LEVELS CHANGED LEVELS CHANGED Up Regulated EarlyResponders Suppression of Transcription Transcripts To: cell, tissue,organ Factors (Level At 4 Hr ≅ 0 Hr) Gluphosinate or plant deathTransporters or Chlorsulfuron following: Change In Cell (Level At 4 Hr >0 Hr) Glyphosate Herbicide Membrane Structure and/or 2, 4-D treatment orKinases And under stress Phosphatases Activation of cell, Germins,Germin- tissue, organ or like proteins, plant death Calcium-bindingfollowing: proteins and H₂O₂ Herbicide generating and treatment or H₂O₂neutralizing under stress proteins. Transcription Activators Change InChromatin Structure And/Or Localized DNA Topology Annexins, cell wallstructural proteins Up Regulated Delayed Responders to Suppression ofTranscription Transcripts Gluphosinate, cell, tissue, organ Factors(Level At 4 Hr < 12 Hr) Chlorsulfuron, or plant death Specific FactorsGlyphosate and/or 2, 4-D following: (Initiation And HerbicideElongation) For treatment or Protein Synthesis under stress Lipidtransfer Activation of cell, proteins tissue, organ orMyrosinase-binding plant death proteins following: Sugar Herbicideinterconverting treatment or enzymes under stress Maintenance Of mRNAStability Maintenance Of Protein Stability Maintenance OfProtein-Protein Interaction Protein translocation factors RNA-bindingproteins Centromere and cytoskeleton proteins Lipases Zn/Cu transportersCell wall structural proteins Down-Regulated Early Responder Suppressionof Transcription Transcripts Repressors Of Stress cell, tissue, organFactors (Level At 0 Hr ≅ 4 Hr) Response State Of or plant death ChangeIn Protein or Metabolism following: Structure By (Level At 0 Hr > 4 Hr)Genes With Herbicide Phosphorylation Discontinued treatment or (Kinases)Or Expression Or UnsTable under stress Dephosphoryaltion mRNA InPresence Of Activation of cell, (Phosphatases) Herbicide or Abiotictissue, organ or Change In Stress plant death Chromatin Structurefollowing: And/Or DNA Herbicide Topology treatment or H₂O₂ neutralizingunder stress proteins Zn/Cu transporters Neutralizing Cell wall proteinsincluding structural proteins SOD and GST Down-Regulated DelayedResponder Suppression of Transcription Transcripts Repressors Of ABAcell, tissue, organ Factors (Level At 4 Hr > 12 Hr) Function State Of orplant death Kinases And Metabolism following: Phosphatases Genes WithHerbicide Stability Of Factors Discontinued treatment or For ProteinExpression Or Unstable under stress Synthesis And mRNA In Presence OfActivation of cell, Degradation herbicide or Abiotic tissue, organ orAmino Acid Stress plant death biosynthesis following: proteins includingHerbicide aspargive synthase treatment or Ca-binding proteins understress Lipid biosynthesis proteins Lipases Zn/Cu transporters Cell wallstructural proteins

While viability modulating polynucleotides and gene products can actalone, combinations of these polynucleotides also affect growth anddevelopment.

Use of Promoters of Viability Genes, Gene Components and Products

Promoters of viability genes can include those that are induced by (1)destructive chemicals, e.g. herbicides, (2) stress, or (3) death. Thesepromoters can be linked operably to achieve expression of anypolynucleotide from any organism. Specific promoters from viabilitygenes can be selected to ensure transcription in the desired tissue ororgan. Proteins expressed under the control of such promoters caninclude those that can induce or accelerate death or those that canprotect plant cells organ death. For example, stress tolerance can beincreased by using promoters of viability genes to drive transcriptionof cold tolerance proteins, for example. Alternatively, promotersinduced by apoptosis can be utilized to drive transcription of antisenseconstructs that inhibit cell death.

III.D.9. Histone Deacetylase (Axel) Responsive Genes, Gene Componentsand Products

The deacetylation of histones is known to play an important role inregulating gene expression at the chromatin level in eukaryotic cells.Histone deacetylation is catalyzed by proteins known as histonedeacetylases (HDAcs). HDAcs are found in multisubunit complexes that arerecruited to specific sites on nuclear DNA thereby affecting chromatinarchitecture and target gene transcription. Mutations in plant HDAcgenes cause alterations in vegetative and reproductive growth thatresult from changes in the expression and activities of HDAc targetgenes or genes whose expression is governed by HDAc target genes. Forexample, transcription factor proteins control whole pathways orsegments of pathways and proteins also control the activity of signaltransduction pathways. Therefore, manipulation of these types of proteinlevels is especially useful for altering phenotypes and biochemicalactivities.

Manipulation of one or more HDAc gene activities is useful to modulatethe biological activities and/or phenotypes listed below. HDAc genes andgene products can act alone or in combination. Useful combinationsinclude HDAc genes and/or gene products with similar biologicalactivities, or members of the same, co-regulated or functionally relatedbiochemical pathways. Such HDAc genes and gene products can function toeither increase or dampen these phenotypes or activities.

Examples of genes whose expression is affected by alterations in HDAcactivity are shown in the Reference and Sequence Tables. These genesand/or gene products are responsible for effects on traits such asinflorescence branching and seed production. They were discovered andcharacterized from a much larger set of genes by experiments designed tofind genes whose mRNA products are affected by a decrease in HDAc geneactivity. These experiments made use of an Arabidopsis mutant havingseverely reduced mRNA levels for the histone deactylase gene AtHDAC1.

Microarray technology allows monitoring of gene expression levels forthousands of genes in a single experiment. This is achieved bysimultaneously hybridizing two differentially labeled fluorescent cDNApools to glass slides that contain spots of DNA (Schena et al. (1995)Science 270: 467-70). The Arabidopsis Functional Genomics Consortium(AFGC) has recently made public the results from such microarrayexperiments conducted with AFGC chips containing 10,000 non-redundantESTs, selected from 37,000 randomly sequenced ESTs generated from mRNAof different tissues and developmental stages.

The sequences of the ESTs showing at least two-fold increases ordecreases over the controls were identified, compared to the Ceresfull-length cDNA and genomic sequence databanks, and identical Ceresclones identified. MA_diff table reports the results of this analysis,indicating those Ceres clones which are up or down regulated overcontrols, thereby indicating the Ceres clones which are HDAc genes. TheMA_diff Table(s) reports the transcript levels of the experiment (seeEXPT ID: Axel (relating to SMD 6654, SMD 6655)). For transcripts thathad higher levels in the samples than the control, a “+” is shown. A “−”is shown for when transcript levels were reduced in root tips ascompared to the control. For more experimental detail see the Examplesection below.

Histone Deacetylase genes are those sequences that showed differentialexpression as compared to controls, namely those sequences identified inthe MA_diff tables with a “+” or “−” indication.

Histone Deacetylase Genes Identified by Cluster Analyses of DifferentialExpression

Histone Deacetylase Genes Identified by Correlation to Genes that areDifferentially Expressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of Histone Deacetylase genes is any group in theMA_clust that comprises a cDNA ID that also appears in Expt ID Axel(relating to SMD 6654, SMD 6655) of the MA_diff table(s).

Histone Deacetylase Genes Identified by Correlation to Genes that CausePhysiological Consequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of HistoneDeacetylase genes. A group in the MA_clust is considered a HistoneDeacetylase pathway or network if the group comprises a cDNA ID thatalso appears in Knock-in or Knock-out tables that causes one or more ofthe phenotypes described in section above.

Histone Deacetylase Genes Identified by Amino Acid Sequence Similarity

Histone Deacetylase genes from other plant species typically encodepolypeptides that share amino acid similarity to the sequences encodedby corn and Arabidopsis Histone Deacetylase genes. Groups of HistoneDeacetylase genes are identified in the Protein Group table. In thistable, any protein group that comprises a peptide ID that corresponds toa cDNA ID member of a Histone Deacetylase pathway or network is a groupof proteins that also exhibits Histone Deacetylase functions/utilities.

III.D.9.a. Use of HDAc Genes, Gene Components and Products to ModulatePhenotypes

HDAc genes and gene products are useful to or modulate one or morephenotypes including growth rate; whole plant, including height,flowering time, etc.; seedling; organ; seed development; embryo;germination, and cell differentiation.

To improve any of the phenotype(s) above, activities of one or more ofthe HDAc genes or gene products can be modulated and tested by screeningfor the desired trait. Specifically, the gene, mRNA levels, or proteinlevels can be altered in a plant utilizing the procedures describedherein and the phenotypes can be assayed. As an example, a plant can betransformed according to Bechtold and Pelletier (1998, Methods. Mol.Biol. 82:259-266) and visually inspected for the desired phenotype ormetabolically and/or functionally assayed according to Wu et al. (2000,Plant J 22: 19-27), Hu et al. (2000, J Biol Chem 275: 15254-64), Johnsonand Turner (1999, Semin Cell Dev Biol 10: 179-88), Koyama et al. (2000,Blood 96: 1490-5), Wu et al. (2000, Plant J 22: 19-27), Li (1999, NatureGenetics 23: 5-6), Adams et al. (2000, Development 127: 2493-2502) andLechner et al. (2000, Biochemistry 39: 1683-92).

III.D.9.b. Use of HDAc Development Genes, Gene Components and Productsto Modulate Biochemical Activities

The activities of one or more of the HDAc genes can be modulated tochange biochemical or metabolic activities and/or pathways such as thosenoted below. Such biological activities can be measured according to thecitations included in the Table below:

BIOCHEMICAL OR METABOLIC ACTIVITIES CITATIONS INCLUDING PROCESS AND/ORPATHWAYS ASSAYS Growth, Differentiation Cell Differentiation Koyama etal. (2000) Blood And Development 96: 1490-5 Cell Cycle Progression Hu etal. (2000) J Biol Chem 275: 15254-64 Metabolism Chromatin Structure Huet al. (2000) J Biol Chem 275: 15254-64 Gene Transcription And Johnsonand Turner (1999) Chromatin Assembly Semin Cell Dev Biol 10: 179-88Reproduction And Seed Seed Development Wu et al. (2000) Plant J 22:19-27 Development Seed Germination Lechner et al. (2000) Biochemistry39: 1683-92 Independent Embryo Ohad et al. (1996) PNAS USA Fertilization93: 5319-24 Fertilization Independent Chaudhury et al. (1997) PNAS SeedDevelopment USA 94: 4222-28 Megagametogenesis Christensen et al. (1997)Sex Plant Reproduc 10: 49-64

Other biological activities that can be modulated by the HDAc genes andgene products are listed in the REFERENCE Table. Assays for detectingsuch biological activities are described in the Protein Domain table.

Profiles of these different HDAc genes are shown in the Table below withexamples of associated biological activities.

EXAMPLES OF TRANSCRIPT PHYSIOLOGICAL BIOCHEMICAL LEVELS TYPE OF GENESCONSEQUENCES ACTIVITY Up Regulated Responders To Gene RepressionTransporters Transcripts HDAc Activity Activity Metabolic enzymes CellCycle Kinases and Progression phosphatases Chromatin TranscriptionCondensation activators Synthesis Of Change in Metabolites chromatinstructure And/Or Proteins and/or localized Modulation Of DNA topologyTransduction Pathways Specific Gene Transcription InitiationDown-Regulated Responder To Hdac Negative Transcription TranscriptsInhibitors Regulation Of factors Genes With Acetylation Change inprotein Discontinued Pathways structure by Expression Or Changes Inphosphorylation UnsTable Mrna In Pathways And (kinases) or Presence OfHdac Processes dephosphorylation Operating In Cells (phosphatases)Changes In Change in Metabolism chromatin structure and/or DNA topologyStability of factors for protein synthesis and degradation Metabolicenzymes

Use of Promoters of Histone Deacetylase Responsive Genes

Promoters of Histone Deacetylase responsive genes are useful fortranscription of any desired polynucleotide or plant or non-plantorigin. Further, any desired sequence can be transcribed in a similartemporal, tissue, or environmentally specific patterns as the HistoneDeacetylase responsive genes where the desired sequence is operablylinked to a promoter of a Histone Deacetylase responsive gene. Theprotein product of such a polynucleotide is usually synthesized in thesame cells, in response to the same stimuli as the protein product ofthe gene from which the promoter was derived. Such promoter are alsouseful to produce antisense mRNAs to down-regulate the product ofproteins, or to produce sense mRNAs to down-regulate mRNAs via sensesuppression.

III.E. Stress Responsive Genes, Gene Components and Products

III.E.1. Cold Responsive Genes, Gene Components and Products

The ability to endure low temperatures and freezing is a majordeterminant of the geographical distribution and productivity ofagricultural crops. Even in areas considered suitable for thecultivation of a given species or cultivar, can give rise to yielddecreases and crop failures as a result of aberrant, freezingtemperatures. Even modest increases (1-2° C.) in the freezing toleranceof certain crop species would have a dramatic impact on agriculturalproductivity in some areas. The development of genotypes with increasedfreezing tolerance would provide a more reliable means to minimize croplosses and diminish the use of energy-costly practices to modify themicroclimate.

Sudden cold temperatures result in modulation of many genes and geneproducts, including promoters. Examples of such cold responsive genesand gene products are shown in the Reference, Sequence, Protein Group,Protein Group Matrix tables, MA_diff and MA_clust tables These genesand/or products are responsible for effects on traits such as plantvigor and seed yield. They were discovered and characterized from a muchlarger set by experiments designed to find genes whose mRNA productschanged in response to cold treatment.

Manipulation of one or more cold responsive gene activities is useful tomodulate the biological activities and/or phenotypes listed below. Coldresponsive genes and gene products can act alone or in combination.Useful combinations include cold responsive genes and/or gene productswith similar transcription profiles, similar biological activities, ormembers of the same or functionally related biochemical pathways. Wholepathways or segments of pathways are controlled by transcription factorproteins and proteins controlling the activity of signal transductionpathways. Therefore, manipulation of the levels of such proteins isespecially useful for altering phenotypes and biochemical activities ofplants. The MA_diff Table(s) reports the transcript levels of theexperiment (see EXPT ID: 108578, 108579, 108533, 108534). Fortranscripts that had higher levels in the samples than the control, a“+” is shown. A “−” is shown for when transcript levels were reduced inroot tips as compared to the control. For more experimental detail seethe Example section below.

Cold genes are those sequences that showed differential expression ascompared to controls, namely those sequences identified in the MA_difftables with a “+” or “−” indication.

Cold Genes Identified by Cluster Analyses of Differential Expression

Cold Genes Identified by Correlation to Genes that are DifferentiallyExpressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of Cold genes is any group in the MA_clust thatcomprises a cDNA ID that also appears in Expt ID 108578, 108579, 108533,108534 of the MA_diff table(s).

Cold Genes Identified by Correlation to Genes that Cause PhysiologicalConsequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of Coldgenes. A group in the MA_clust is considered a Cold pathway or networkif the group comprises a cDNA ID that also appears in Knock-in orKnock-out tables that causes one or more of the phenotypes described insection above.

Cold Genes Identified by Amino Acid Sequence Similarity

Cold genes from other plant species typically encode polypeptides thatshare amino acid similarity to the sequences encoded by corn andArabidopsis Cold genes. Groups of Cold genes are identified in theProtein Group table. In this table, any protein group that comprises apeptide ID that corresponds to a cDNA ID member of a Cold pathway ornetwork is a group of proteins that also exhibits Coldfunctions/utilities.

Such cold responsive genes and their products can function to eitherincrease or dampen the phenotypes and activities below either inresponse to cold treatment or in the absence of cold temperaturefluctuations.

Further, promoters of cold responsive genes, as described in theReference tables, for example, are useful to modulate transcription thatis induced by ABA or any of the following phenotypes or biologicalactivities below.

III.E.1.a. Use of Cold-Responsive Genes to Modulate Phenotypes

Cold responsive genes and gene products are useful to or modulate one ormore phenotypes including cold tolerance, below 7° C., for example,cells, organelles, proteins, dehydration resistance, growth rate, wholeplant, including height, bolting time, etc., organs, biomass, fresh anddry weight during any time in plant life, such as maturation, number,size, and/or weight of flowers, seeds, branches, or leaves; seed yieldin terms of number, size, weight, harvest index, or water content, fruityield in terms of number, size, weight, harvest index, water content.

To regulate any of the phenotype(s) above, activities of one or more ofthe cold responsive genes or gene products can be modulated and theplants can be tested by screening for the desired trait. Specifically,the gene, mRNA levels, or protein levels can be altered in a plantutilizing the procedures described herein and the phenotypes can bescreened for variants as in Winkler et al. (1998) Plant Physiol 118:743-50 and assayed, for example, in accordance to Steponokus et al.(1993) Biochimica et Biophysica Acta 1145: 93-104; Quinn (1988) SympSoc. Exp. Biol. 42: 237-258; Bectold and Pelletier (1998) Methods Mol.Biol. 82: 259-266; Kasuga et al. (1999) Nature Biotechnology 17:287-291; Guy et al. (1998) Cryobiology 36: 301-314; or Liu et al. (1998)Plant Cell 10: 1391-1406.

III.E.1.b. Use of Cold-Responsive Genes to Modulate BiochemicalActivities

The activities of one or more of the cold responsive genes can bemodulated to change biochemical or metabolic activities and/or pathwayssuch as those noted below. Such biological activities are documented andcan be measured according to the citations above and those included inthe Table below:

BIOCHEMICAL OR METABOLIC ACTIVITIES PROCESS AND/OR PATHWAYS CITATIONSINCLUDING ASSAYS Cold Tolerance Viability Of Plant Steponkus (1998) PNASUSA 95: Protoplasts At Low 14570-14575 Temperatures. Viability Of YeastAt Low Schirmer et al. (1994) Plant Cell 6: Temperatures. 1899-1909Complementation Of Yeast Zentella et al. (1999) Plant Tsp MutantPhysiology, 119: 1473-1482 Viability Of E. Coli At Low Yeh et. al.(1997) PNAS 94: 10967-10972 Temperatures. Induction Of Cold Shock Pearce(1999) Plant Growth Response Genes Regulation 29: 47-76. LipidComposition Altered Composition Of Sayanova et al. (1999) Journal ofMembrane Fatty Acids Experimental Botany 50: 1647-1652 Sayanova (1997)PNAS USA 94: 4211-4216 ALTERATION OF Porta et al. (1999) Plant and CellLIPOXYGENASE Physiology 40: 850-858. ENZYME ACCUMULATION AND ACTIVITYProtein PROTEIN Wisniewski et al.(1999) Physiologia CompositionDENATURATION Plantarum 105: 600-608 Protein Hydrophilicity Steponkus(1998) PNAS USA 95: 14570-14575 Modulation of Induced TranscriptionCurrent Protocols in Molecular Transcription Factors And Other DnaBiology/edited by Frederick M. Induced by Low Binding Proteins Ausubel..[et al.]. New York: Temperatures Transcription Of Published by GreenePub. Associates Specific Genes and Wiley-Interscience: J. Wiley, c1987.Steponkus (1998) PNAS USA 95: 14570-14575 Kadyrzhanova et al., Plant MolBiol (1998) 36(6): 885-895; and Pearce et al., Plant Physiol (1998)117(3): 787-795 Signal Plasma Membrane Goodwin et al., Plant Mol Biol(1996) Transduction Proteins 31(4) 777-781; and Koike et al., Plant CellPhysiol (1997) 38(6): 707-716 Oxygen Glutathione Kocsy et al., Planta(2000) 210(2): Scavengers 295-301 Accumulation Active O₂ Tao et al.,Cryobiology (1998) and H₂O₂ Scavengers 37(1): 38-45 Dehydration DehydrinIsmail et al., Plant Physiol (1999) 120(1): 237-244 Transcription ofmRNA Kaye et al., Plant Physiol (1998) 116(4): 1367-1377 MetabolismSoluble Sugars and/or Wanner et al., (1999) Plant Physiol Proline120(2): 391-400 RNA/DNA Stabilization of Jiang, Weining et al., (1997)Journal of Chaperone RNA/DNA through Biological Chemistry, 272: 196-202.RNA binding and Fukunaga et al., (1999) Journal of modulation of RNAPlant Research, 112: 263-272. translation through RNA binding and orunwinding. Protein Chaperone Stabilize protein Forreiter and Nover(1998) Journal of structure and facilitate Biosciences 23: 287-302protein folding

Other biological activities that can be modulated by the cold responsivegenes and their products are listed in the Reference tables. Assays fordetecting such biological activities are described in the Protein Domaintable.

Cold responsive genes are characteristically differentially expressed inresponse to fluctuating cold temperature levels, whether internal orexternal to an organism or cell. The MA_diff table reports the changesin transcript levels of various cold responsive genes in the aerialparts of seedlings at 1 and 6 hours at 4° C. in the dark as compared toaerial parts of seedlings covered with aluminium foil, and grown at 20°C. in the growth chamber.

The data from this time course can be used to identify a number of typesof cold responsive genes and gene products, including “early responders”and “delayed responders”. Profiles of these different cold responsivegenes are shown in the Table below together with examples of the kindsof associated biological activities.

EXAMPLES OF GENE FUNCTIONAL TYPE OF BIOCHEMICAL EXPRESSION CATEGORY OFBIOLOGICAL ACTIVITIES OF GENE LEVELS GENE ACTIVITY PRODUCTS UpregulatedGenes Early Perception Of Transcription Factors (Level At 1 h ≅ 6 h)Responders To Cold Kinases And or Cold Induction Of Phosphatases (LevelAt 1 h > 6 h) Cold Response Amino Acid Sugar And Signal MetaboliteTransporters Transduction Carbohydrate Catabolic Pathways And AnabolicEnzymes. Initiating Lipid Biosynthesis Specific Gene EnzymesTranscription Lipid Modification Osmotic Enzymes, Example AdjustmentDesaturases Alteration Of Ice Crystal Binding Lipid ProteinsComposition. Hydrophilic Proteins Ice Nucleation Inhibition MitigationOf Dehydration By Sequestering Water Stress Response Repression OfTranscription Factors General Kinases And Biochemical PhosphatasesPathways To Protein Stability Factors Optimize Cold mRNA StabilityFactors Response mRNA Translation Pathways. Factors Stabilization OfProtein Turnover Factors Protein/Enzyme Oxygen Radical Activity At LowScavengers, Example- Temperature Peroxidases Protection EnergyGeneration Against Oxidative Enzymes EtOH Stress DetoxificationAnaerobic Metabolism Upregulated Genes Delayed Respiration,Transcription Factors (Level At 1 h < 6 h) Responders To PhotosynthesisKinases And Cold Stress And Protein Phosphatases Cold Synthesis ProteinStability Factors Acclimation Carbohydrate mRNA Stability Factors GenesAnd Amino Acid mRNA Translation Solute Factors Accumulation ProteinTurnover Factors Increased Fatty Oxygen Radical Acid DesaturationScavengers, Peroxidase To Increase Lipid Metabolic Enzymes MembraneStability Increased Accumulation Or Activity Of Oxidative StressProtection Proteins Stabilization Of Protein/Enzyme Activity At LowTemperature Protection Against Oxidative Stress Extracellular MatrixModification Stress Response Stabilization Of Transcription FactorsGenes Protein/Enzyme Kinases And Activity At Low PhosphatasesTemperature Protein Stability Factors Protection mRNA Stability FactorsAgainst Oxidative mRNA Translation Stress Factors Anaerobic ProteinTurnover Factors Metabolism Oxygen Radical Scavengers, Example-Peroxidase Energy Generation Enzymes, Etoh Detoxification DownregulatedEarly Negative Transcription Factors (Level At 1 h ≅ 6 h) ResponderRegulation Of Kinases And (Level At 6 h > 1 h) Repressors Of Cold SignalPhosphatases Cold Stress Transduction Protein Stability FactorsMetabolism Pathways Released mRNA Stability Factors mRNA TranslationFactors Protein Turnover Factors Genes With Negative Cold RepressedDiscontinued Regulation Of Metabolic Pathway Expression Or Cold InducedProteins UnsTable Transcription Factors Coordinating And mRNA In ColdReduced Controlling Central C and Reduction In N Metabolism GeneExpression Storage Proteins In Pathways Not Required Under ColdConditions Induced mRNA Turnover Down-Regulated Delayed Maintenance OfTranscription Factors Transcripts Responder Cold Induced State KinasesAnd (Level At 1 h > 6 h) Repressors Of Of Metabolism Phosphatases ColdStress Reduction In Protein Stability Factors Metabolism Gene ExpressionmRNA Stability Factors Genes With For Pathways Not mRNA TranslationDiscontinued Required Under Factors Expression Or Cold ConditionsProtein Turnover Factors UnsTable Induced mRNA Cold Repressed mRNA InCold Turnover Metabolic Pathway Proteins Factors Coordinating AndControlling Central C and N Metabolism Storage Proteins

Further, any desired sequence can be transcribed in similar temporal,tissue, or environmentally specific patterns as the cold responsivegenes when the desired sequence is operably linked to a promoter of acold responsive gene.

III.E.2. Heat Responsive Genes, Gene Components and Products

The ability to endure high temperatures is a major determinant of thegeographical distribution and productivity of agricultural crops.Decreases in yield and crop failure frequently occur as a result ofaberrant, hot conditions even in areas considered suitable for thecultivation of a given species or cultivar. Only modest increases in theheat tolerance of crop species would have a dramatic impact onagricultural productivity. The development of genotypes with increasedheat tolerance would provide a more reliable means to minimize croplosses and diminish the use of energy-costly practices to modify themicroclimate.

Changes in temperature in the surrounding environment or in a plantmicroclimate results in modulation of many genes and gene products.Examples of such heat stress responsive genes and gene products areshown in the Reference, Sequence, Protein Group, Protein Group Matrix,MA_diff and MA_clust tables. These genes and/or products are responsiblefor effects on traits such as plant vigor and seed yield. They werediscovered and characterized from a much larger set by experimentsdesigned to find genes whose mRNA products changed in response to hightemperatures.

While heat stress responsive polynucleotides and gene products can actalone, combinations of these polynucleotides also affect growth anddevelopment. Useful combinations include different heat stressresponsive polynucleotides and/or gene products that have similartranscription profiles or similar biological activities, and members ofthe same or similar biochemical pathways. Whole pathways or segments ofpathways are controlled by transcription factor proteins and proteinscontrolling the activity of signal transduction pathways. Therefore,manipulation of such protein levels is especially useful for alteringphenotypes and biochemical activities of plants. In addition, thecombination of a heat stress responsive polynucleotide and/or geneproduct with other environmentally responsive polynucleotide is alsouseful because of the interactions that exist between stress pathways,pathogen stimulated pathways, hormone-regulated pathways, nutritionalpathways and development. Here, in addition to polynucleotides havingsimilar transcription profiles and/or biological activities, usefulcombinations include polynucleotides that may have differenttranscription profiles, but which participate in common or overlappingpathways. The MA_diff Table(s) reports the transcript levels of theexperiment (see EXPT ID: 108576, 108577, 108522, 108523). Fortranscripts that had higher levels in the samples than the control, a“+” is shown. A “−” is shown for when transcript levels were reduced inroot tips as compared to the control. For more experimental detail seethe Example section below.

Heat genes are those sequences that showed differential expression ascompared to controls, namely those sequences identified in the MA_difftables with a “+” or “−” indication.

Heat Genes Identified by Cluster Analyses of Differential Expression

Heat Genes Identified by Correlation to Genes that are DifferentiallyExpressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of Heat genes is any group in the MA_clust thatcomprises a cDNA ID that also appears in Expt ID 108576, 108577, 108522,108523 of the MA_diff table(s).

Heat Genes Identified by Correlation to Genes that Cause PhysiologicalConsequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of Heatgenes. A group in the MA_clust is considered a Heat pathway or networkif the group comprises a cDNA ID that also appears in Knock-in orKnock-out tables that causes one or more of the phenotypes described insection above.

Heat Genes Identified by Amino Acid Sequence Similarity

Heat genes from other plant species typically encode polypeptides thatshare amino acid similarity to the sequences encoded by corn andArabidopsis Heat genes. Groups of Heat genes are identified in theProtein Group table. In this table, any protein group that comprises apeptide ID that corresponds to a cDNA ID member of a Heat pathway ornetwork is a group of proteins that also exhibits Heatfunctions/utilities.

Such heat stress responsive genes and gene products can function eitherto increase or dampen the above phenotypes or activities either inresponse to changes in temperature or in the absence of temperaturefluctuations.

Further, promoters of heat responsive genes, as described in theReference tables, for example, are useful to modulate transcription thatis induced by heat or any of the following phenotypes or biologicalactivities below.

III.E.2.a. Use of Heat Stress Responsive Genes to Modulate Phenotypes

Heat stress responsive genes and gene products can be used to alter ormodulate one or more phenotypes including heat tolerance (above 20° C.,23° C., 27° C., 30° C., 33° C., 37° C., 40° C. or 42° C.), heattolerance of cells, of organelles, of proteins, of cells or organellesdehydration resistance, growth rate, whole plant, including height,bolting time, etc., organs, biomass, fresh and dry weight during anytime in plant life, such as maturation, number, size, and weight offlowers, seeds, branches, or leaves; seed yield in number, size, weight,harvest index; fruit yield in terms of number, size, weight, or harvestindex, stress responses such as mediation of response to desiccation,drought, salt, disease, wounding, cold and other stresses, andreproduction

To regulate any of the phenotype(s) above, activity of one or more ofthe heat stress responsive genes or gene products can be modulated andthe plants tested by screening for the desired trait. Specifically, thegene, mRNA levels, or protein levels can be altered in a plant utilizingthe procedures described herein and the phenotypes can be assayed. As anexample, a plant can be transformed according to Bechtold and Pelletier(1998, Methods. Mol. Biol. 82:259-266) and/or screened for variants asin Winkler et al. (1998) Plant Physiol 118: 743-50 and visuallyinspected for the desired phenotype or metabolically and/or functionallyassayed according to Queitsch et al. (2000, The Plant Cell 12: 479-92).

III.E.2.b. Use of Heat Stress Responsive Genes to Modulate BiochemicalActivities

The activities of one or more of the heat stress responsive genes can bemodulated to change biochemical or metabolic activities and/or pathwayssuch as those noted below. Such biological activities can be measuredaccording to the citations included in the Table below:

BIOCHEMICAL OR METABOLIC ACTIVITIES CITATION INCLUDING PROCESS AND/ORPATHWAYS ASSAY Cell Growth and Regulation And Wisniewski et al.Differentiation Molecular Chaperones (1999) Physiolgia Maintenance OfNative Plantarum 105: 600-608 Conformation (Cytosolic Queitsch et al.Proteins) (2000) The Plant Reactivation Of Cell 12: 479-92 AggregationAnd Protein Lee and Vierling Folding (2000) Plant Autoregulation Of HeatPhysiol. 122: 189-197 Shock Response Schwechheimer Regulation Of (1998)Plant Mol Translational Efficiency Biol 36: 195-204 Regulation Of KinaseShi et al. (1998) Activity Genes and Regulation Of Calcium Development12: Mediated Signal 654-66 Transduction Wells et al. (1998) Genes andDevelopment 12: 3236-51 Lis et al. (2000) Genes and Development 14:792-803 Malho, R.(1999) Plant Biology 1: 487-494. Sheen, Jen.(1996)Science 274: 1900-1902. Farmer, P. et al., (1999.) Biochimica etBiophysica Acta 1434: 6-17. Gene regulation Transcriptional RegulationOf Current Protocols in Heat Induced Proteins Molecular Biology/editedThrough DNA Binding by Frederick M. Ausubel.. Proteins. [et al.]. NewYork: Transcriptional Regulation Of Published by Greene Pub. HeatInduced Proteins Associates and Wiley- Through Protein-ProteinInterscience: J. Wiley, Interactions Between DNA c1987. Binding ProteinsAnd Steponkus (1998) Coactivators. PNAS USA 95: TranscriptionalRegulation Of 14570-14575 Heat Induced Proteins Gubler et al. ThroughProtein (1999) Plant Phosphorylation And Journal 17: 1-9Dephosphorylation Glenn et al. Transcriptional Regulation Of (1999)Journal of Thermal Stress Induced Biological Genes By Protein-ProteinChemistry, 274: Interactions. 36159-36167 Translational Regulation OfZhou et al., (1997) Thermal Stress Induced EMBO Journal16: 3207-3218.Messenger Rnas. Sessa et al., Transcriptional Regulation Of (2000) EMBOJournal 19: Heat Induced Genes Through 2257-2269. Chromatin Remodeling.Burnett et al., (2000) Journal of Experimental Botany. 51: 197-205.Osterlund et al., (2000) Nature 405: 462-466. Gross and Watson (1998)Canadian Journal of Microbiology, 44: 341-350 Luo, R. X., Dean, D. C.(1999) Journal of the National Cancer Institute 91: 1288-1294. Chromatinprotocols (1999) edited by Peter B. Becker. Totowa, N. J.: Humana Press.Cell Structure Thermal Stress Protection By Goodwin et al. (1996) PlasmaMembrane Anchored Plant Mol Biol 31(4) 777-781; Or Secreted And/Or Celland Wall Associated Proteins. Koike et al. (1997) Plant Cell Physiol38(6): 707-716 Signal Transduction Regulation Of Thermal Stress Jonak(1996) Proceedings Pathways And Protein of the National Academy ofActivity By Protein Kinase Sciences of the United And ProteinPhosphatase States of America, 93: Mediated Phosphorylation 11274-11279.And Dephosphorylation Monroy. et al., (1998) Respectively. AnalyticalBiochemistry 265: 183-185. Photosynthesis Regulation Of Schroda et al.(1999) The Photoprotection And Repair Plant Cell 11: 1165-178 OfPhotosystem II Oh and Lee (1996) J Plant Biol. 39: 301-07 StressResponse Regulation Of Cytosol Dat et al. (1998) Plant Peroxide LevelsPhysiol 116: 1351-1357 Regulation Of Heat Shock Kurek et al. (1999)Plant Factor Binding Physiol 119: 693-703 Regulation Of ProteinStorozhenko et al. (1998) Stability During Thermal Plant Physiol 118:1005-14 Stress Soto et al. (1999) Plant Nucleocytoplasmic Export OfPhysiol 120: 521-28 Heat Shock Protein Mrnas Yeh et al. (1997) PNAS 94:Regulation/Reconfiguration 10967-10972 Of Cell Architecture Winkler etal. (1998) Plant Regulation Of Pathways For Physiol 118: 743-50Reactivation Of “Damaged” Saavedra et al. (1997) And/Or DenaturedProteins Genes and Development Regulation Of Protein 11: 2845-2856Degradation During Thermal Parsell and Lindquist Stress. (1993). Ann.Rev. Genet. Regulation Of Osmotic 27: 437-496. Potential During ThermalParsell and Lindquist Stress. (1993). Ann. Rev. Genet. Regulation OfUniversal 27: 437-496. Stress Protein Homologue Georgopoulos and WelchActivity By Phosphorylation (1993). Ann Rev. Cell Biol. AndDephosphorylation. 9: 601-634. Regulation Of Dehydrin, Vierstra, RichardD. LEA-Like And Other Heat (1996) Plant Molecular STable ProteinAccumulation Biology, 32: 275-302. Vierstra, Richard D.; Callis, Judy.(1999) Plant Molecular Biology, 41: 435-442. Liu, J. et al., (1998)PlantScience 134: 11-20. Freestone, P. 1997et al., Journal of MolecularBiology, v. 274: 318-324. Robertson, A. J. (1994) Plant Physiology 105:181-190.

Other biological activities that can be modulated by the heat stressresponsive genes and gene products are listed in the Reference tables.Assays for detecting such biological activities are described in theProtein Domain table.

Heat stress responsive genes are characteristically differentiallytranscribed in response to fluctuating temperatures, whether internal orexternal to an organism or cell. The MA_diff table reports the changesin transcript levels of various heat stress responsive genes in aerialtissues at 1 and 6 hours after plants were placed at 42° C. as comparedto aerial tissues kept at 20° C. growth chamber temperature.

The data from this time course can be used to identify a number of typesof heat stress responsive genes and gene products, including “earlyresponders to heat stress,” “delayed responders to heat stress,” “earlyresponder repressors,” and “delayed repressor responders.” Profiles ofthese different heat stress responsive genes are shown in the Tablebelow together with examples of the kinds of associated biologicalactivities.

EXAMPLES OF GENE FUNCTIONAL BIOCHEMICAL EXPRESSION CATEGORY OFPHYSIOLOGICAL ACTIVITIES/GENE LEVELS GENE CONSEQUENCES PRODUCTS UpRegulated Early Responders Heat Stress Perception TranscriptionTranscripts To Heat Stress Modulation Of Heat Factors (Level At 1 h ≅ 6h) Stress Response Transporters Or Transduction Changes In Cell (LevelAt 1 h > 6 h) Pathways Membrane Structure Specific Gene Kinases AndTranscription Phosphatases Initiation Transcription Conditional Shift InActivators Preferential Changes In Translation Of Chromatin StructureTranscripts And/Or Localized Changes In Cell Dna Topology ArchitectureTo Modification Of Pre- Optimize Cell Existing Translation Adaptation ToHeat Factors By Stress Phosphorylation (Kinases) Or Dephosphorylation(Phosphatases) Synthesis Of New Translation Factors Stability OfMediators Of Protein-Protein Interaction Heat Shock Proteins Changes InOrganelle Structures, Membranes And Energy-Related Activities ProteinsTo Catalyse Metabolic Turnover Up Regulated “Delayed” Maintenance OfTranscription Transcripts Responders Response To Heat Factors (Level At1 h < 6 h) Maintenance Of Stress Specific Factors Heat StressMaintenance Of (Initiation And Response Protein Stability AndElongation) For Conformation Protein Synthesis Maintenance Of MrnaStability Heat Shock Proteins Changes In Organelle Structures, MembranesAnd Energy-Related Activities Proteins To Catalyse Metabolic Turnover.Stability Of Mediators Of Protein-Protein Interaction Down-RegulatedEarly Responder Negative Regulation Transcription Transcripts RepressorsOf Of Heat Stress Factors And (Level At 1 h ≅ 6 h) “Normal” State OfResponse Released Activators Or Metabolism Changes In Change In Protein(Level At 6 h > 1 h) Genes With Biochemical And Structure ByDiscontinued Signal Transduction Phosphorylation Expression Or PathwaysAnd (Kinases) Or UnsTable mRNA Processes Operating In DephosphoryaltionIn Presence Of Cells (Phosphatases) Heat Stress Reorientation Of ChangeIn Metabolism Chromatin Structure And/Or Dna Topology Down-RegulatedDelayed Maintenance Of Heat Transcription Transcripts Repressors OfStress Response Factors And (Level At 1 hr > “Normal” State OfMaintenance Of Activators 6 hr) Metabolism Pathways Released Kinases AndGenes With From Repression Phosphatases Discontinued Changes In PathwaysStability Of Factors Expression Or And Processes For Protein UnsTablemRNA Operating In Cells Translation In Presence Of Reorientation Of HeatStress Metabolism

Further, any desired sequence can be transcribed in similar temporal,tissue, or environmentally specific patterns as the heat responsivegenes when the desired sequence is operably linked to a promoter of aheat responsive gene.

III.E.3. Drought Responsive Genes, Gene Components and Products

The ability to endure drought conditions is a major determinant of thegeographical distribution and productivity of agricultural crops.Decreases in yield and crop failure frequently occur as a result ofaberrant, drought conditions even in areas considered suitable for thecultivation of a given species or cultivar. Only modest increases in thedrought tolerance of crop species would have a dramatic impact onagricultural productivity. The development of genotypes with increaseddrought tolerance would provide a more reliable means to minimize croplosses and diminish the use of energy-costly practices to modify themicroclimate.

Drought conditions in the surrounding environment or within a plant,results in modulation of many genes and gene products. Examples of suchdrought responsive genes and gene products are shown in the Referenceand Sequence Tables. These genes and/or products are responsible foreffects on traits such as plant vigor and seed yield. They werediscovered and characterized from a much larger set by experimentsdesigned to find genes whose mRNA products changed in response toavailability of water.

While drought responsive polynucleotides and gene products can actalone, combinations of these polynucleotides also affect growth anddevelopment. Useful combinations include different drought responsivepolynucleotides and/or gene products that have similar transcriptionprofiles or similar biological activities, and members of the same orsimilar biochemical pathways. Whole pathways, or segments of pathwaysare controlled by transcription factor proteins and proteins controllingthe activity of signal transduction pathways. Therefore, manipulation ofthe levels of such proteins is especially useful for altering phenotypesand biochemical activities of plants. In addition, the combination of adrought responsive polynucleotide and/or gene product with anotherenvironmentally responsive polynucleotide is also useful because of theinteractions that exist between hormone-regulated pathways, stresspathways, nutritional pathways and development. Here, in addition topolynucleotides having similar transcription profiles and/or biologicalactivities, useful combinations include polynucleotides that may havedifferent transcription profiles but which participate in a commonpathway. The MA_diff Table(s) reports the transcript levels of theexperiment (see EXPT ID: 108572, 108573, 108502, 108503, 108504, 108556,108482, 108483, 108473, 108474, 108477). For transcripts that had higherlevels in the samples than the control, a “+” is shown. A “−” is shownfor when transcript levels were reduced in root tips as compared to thecontrol. For more experimental detail see the Example section below.

Drought genes are those sequences that showed differential expression ascompared to controls, namely those sequences identified in the MA_difftables with a “+” or “−” indication.

Drought Genes Identified by Cluster Analyses of Differential Expression

Drought Genes Identified by Correlation to Genes that are DifferentiallyExpressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of Drought genes is any group in the MA_clust thatcomprises a cDNA ID that also appears in Expt ID 108572, 108573, 108502,108503, 108504, 108556, 108482, 108483, 108473, 108474, 108477 of theMA_diff table(s).

Drought Genes Identified by Correlation to Genes that CausePhysiological Consequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of Droughtgenes. A group in the MA_clust is considered a Drought pathway ornetwork if the group comprises a cDNA ID that also appears in Knock-inor Knock-out tables that causes one or more of the phenotypes describedin section above.

Drought Genes Identified by Amino Acid Sequence Similarity

Drought genes from other plant species typically encode polypeptidesthat share amino acid similarity to the sequences encoded by corn andArabidopsis Drought genes. Groups of Drought genes are identified in theProtein Group table. In this table, any protein group that comprises apeptide ID that corresponds to a cDNA ID member of a Drought pathway ornetwork is a group of proteins that also exhibits Droughtfunctions/utilities.

Such drought responsive genes and gene products can function to eitherincrease or dampen the above phenotypes or activities either in responseto drought conditions or in the absence of drought conditions. Further,promoters of drought responsive genes, as described in the Referencetables, for example, are useful to modulate transcription that isinduced by drought or any of the following phenotypes or biologicalactivities below.

More specifically, drought responsive genes and gene products are usefulto or modulate one or more phenotypes including growth, roots, stems,buds, leaves, development, cell growth, leaves, fruit development, seeddevelopment, senescence, stress responses, and mediates response todesiccation, drought, salt and cold.

Further, any desired sequence can be transcribed in similar temporal,tissue, or environmentally specific patterns as the drought responsivegenes when the desired sequence is operably linked to a promoter of adrought responsive gene.

To produce the desired phenotype(s) above, one or more of the droughtresponse genes or gene products can be tested by screening for thedesired trait. Specifically, the gene, mRNA levels, or protein levelscan be altered in a plant utilizing the procedures described herein andthe phenotypes can be assayed. As an example, a plant can be transformedaccording to Bechtold and Pelletier (1998, Methods. Mol. Biol.82:259-266) and/or screened for variants as in Winkler et al. (1998)Plant Physiol 118: 743-50 and visually inspected for the desiredphenotype or metabolically and/or functionally assayed according toRuzin (1999, In: Plant Microtechnique and Microscopy, Oxford UniversityPress, London) and Khanna-Chopra et al. (1999, BBRC 255:324-7).

Alternatively, the activities of one or more of the drought responsivegenes can be modulated to change biochemical or metabolic activitiesand/or pathways such as those noted below. Such biological activitiescan be measured according to the citations included in the Table below:

BIOCHEMICAL OR METABOLIC ACTIVITIES GENERAL CATEGORY AND/OR PATHWAYSASSAY Cell Growth and Preservation of Leaf Sub-Cellular Jagtap et al.(1998) J Exptl Differentiation Structures Including Botany 49: 1715-1721Photosynthetic Apparatus Preservation of Cell Membrane Munne-Bosch andAlegre Structures (2000) Planta 210: 925-31 Regulation of Stomatal Menkeet al. (2000) Plant development and Physiology Physiol. 122: 677-686.Regulation of Factors Involved in Harrak et al. (1999) Plant theDrought-adapted change in Physiol. 121: 557-564. cell ultrastructurePhysiology Modulation of Transpiration Allen et al. (1999) Plant Cell11: 1785-98 Li et al. (2000) Science 287: 300-303 Burnett et al. (2000)J Exptl Bot 51: 197-205 Raschke (1987) In: Stomatal function, Zeiger etal., Eds, 253-79 Modulation of Photosynthesis Sung and Krieg (1979)Plant Physiol 64: 852-56 Regulation of Epicuticular Wax Rhee et al.(1998) Plant Biosynthesis Physiol 116: 901-11 Regulation of CarotenoidAlegre (2000) Planta 210: Biosynthesis 925-31 Loggini et al (2000) PlantPhysiol 119: 1091 Stress Response Modulation of Leaf Rolling to Taiz andZeiger (1991) In: minimize water loss Plant Physiology,Benjamin/Cummings Publishing Co., Redwood City, pp 346-70 Modulation ofOsmolite Hare et al. (1998) Plant, Synthesis Cell and Environment 21:535-553 Huan et al. (2000) Plant Physiol 122: 747-756 Regulation of geneHare et al. (1999) J. Exptl. transcriptional activity specific to Botany333: 413-434. the establishment of drought tolerance Regulation ofprotein degradation Lee and Vierling (2000) and reactivation duringdrought Plant Physiol. 122: 189-197 stress conditionModulation/reconfiguration of Lis et al. (2000) Genes and translationmachineries Development 14: 792-803 (“recycling” mechanisms) adapTableto drought condition Signal Transduction Regulation of Ion SequestrationBush and Jones (1987) Cell Calcium 8: 455-72 Regulation of NuclearTargeted Ferringno and Silver Protein Transport (1999) Methods in CellBiology 58: 107-22 Regulation of Cytoplasmic Ca+2 Shi et al. (1999)Plant Cell 11: 2393-2406 Regulation of Kinase Synthesis Li et al. (2000)Science and Activity 287-300-03 Modulation of Molecular Mayhew et al(1996) Chaperone Activity Nature 379: 420-26 Kimura et al. (1995)Science 268: 1362-1365.

Other biological activities that can be modulated by the droughtresponsive genes and gene products are listed in the Reference Tables.Assays for detecting such biological activities are described in theProtein Domain table.

Drought responsive genes are characteristically differentiallytranscribed in response to drought conditions, whether internal orexternal to an organism or cell. The MA_diff table(s) report(s) thechanges in transcript levels of various drought responsive genes at 1and 6 hours after aerial tissues were isolated and left uncovered atroom temperature on 3 MM paper, as compared to isolated aerial tissuesplaced on 3 MM paper wetted with Hoagland's solution. The data from thistime course can be used to identify a number of types of droughtresponsive genes and gene products, including “early responders,” and“delayed responders.” Profiles of these different drought responsivegenes are shown in the Table below together with examples of the kindsof associated biological activities.

EXAMPLES OF GENE FUNCTIONAL BIOCHEMICAL EXPRESSION CATEGORY OFPHYSIOLOGICAL ACTIVITIES OF GENE LEVELS GENE CONSEQUENCES PRODUCTS Upregulated Early responders to Drought perception Transcription factorstranscripts drought leading to the Transporters (level at 1 hr ≈ 6 hr)establishment of (level at 1 hr > 6 hr) tolerance to drought Modulationof drought Change in cell membrane response transduction structurepathways Kinases and phosphatases Specific gene Transcription activatorstranscription initiation Change in chromatin structure and/or localizedDNA topology Conditional shift in Modification of pre- preferentialtranslation existing translation factors of transcripts byphosphorylation (kinases) or dephosphorylation (phosphatases) Synthesisof new translation factors Changes in cell Stability of mediators ofarchitecture to optimize protein-protein interaction cell adaptation toheat stress Changes in cell Synthesis and/or stability division cycle offactors regulating cell division Up regulated Maintenance of Maintenanceof Transcription factors transcripts drought response response todrought and Specific factors (initiation (level at 1 hr < 6 hr)“Delayed” responders maintenance of and elongation) for proteindrought-tolerance synthesis mechanisms RNA-binding proteins effectivefor mRNA stability Change in chromatin structure and/or DNA topologyMaintenance of Stability of mediators of mechanisms effectiveprotein-protein interaction for ions sequestration, Stability of factorsto osmolite biosynthesis, effectively utilize pre- nuclear proteinexisting translation transport, regulation of machinery (“recycling”cytoplasmic Ca+2, and mechanisms) under regulation of proteins droughtcondition effective for maintaining protein stability and conformationMaintenance of cellular Stability of mediators of structuresprotein-protein interaction Down-regulated Early responder Negativeregulation of Transcription factors and transcripts repressors of“normal” drought response activators (level at 1 hr ≈ 6 hr) state ofmetabolism inducible pathways Change in protein structure (level at 6hr > 1 hr) Genes with released by phosphorylation discontinued Changesin (kinases) or expression or biochemical and signal dephosphoryaltionunsTable mRNA in transduction pathways (phosphatases) presence of waterand processes operating Change in chromatin stress in cells structureand/or DNA topology Down-regulated Delayed repressors of Maintenance ofTranscription factors and transcripts “normal” state of drought responseactivators (level at 1 hr > 6 hr) metabolism Maintenance of Kinases andphosphatases Genes with pathways released from Stability of factors fordiscontinued repression protein translation expression or Changes inpathways unsTable mRNA in and processes operating presence of water incells stress

Use of Promoters of Drought Responsive Genes

Promoters of Drought responsive genes are useful for transcription ofany desired polynucleotide or plant or non-plant origin. Further, anydesired sequence can be transcribed in a similar temporal, tissue, orenvironmentally specific patterns as the Drought responsive genes wherethe desired sequence is operably linked to a promoter of a Droughtresponsive gene. The protein product of such a polynucleotide is usuallysynthesized in the same cells, in response to the same stimuli as theprotein product of the gene from which the promoter was derived. Suchpromoter are also useful to produce antisense mRNAs to down-regulate theproduct of proteins, or to produce sense mRNAs to down-regulate mRNAsvia sense suppression.

III.E.4. Wounding Responsive Genes, Gene Components and Products

Plants are continuously subjected to various forms of wounding fromphysical attacks including the damage created by pathogens and pests,wind, and contact with other objects. Therefore, survival andagricultural yields depend on constraining the damage created by thewounding process and inducing defense mechanisms against future damage.

Plants have evolved complex systems to minimize and/or repair localdamage and to minimize subsequent attacks by pathogens or pests or theireffects. These involve stimulation of cell division and cell elongationto repair tissues, induction of programmed cell death to isolate thedamage caused mechanically and by invading pests and pathogens, andinduction of long-range signaling systems to induce protectingmolecules, in case of future attack. The genetic and biochemical systemsassociated with responses to wounding are connected with thoseassociated with other stresses such as pathogen attack and drought.

Wounding results in the modulation of activities of specific genes and,in consequence, of the levels of key proteins and metabolites. Thesegenes, called here wounding responsive genes, are important forminimizing the damage induced by wounding from pests, pathogens andother objects. Examples of such wounding responsive genes, genecomponents and products are shown in the Reference, Sequence, ProteinGroup, Protein Group Matrix, MA_diff, and MA_clust tables. They can beactive in all parts of a plant and so where, when and to what extentthey are active is crucial for agricultural performance and for thequality, visual and otherwise, of harvested products. They werediscovered and characterized from a much larger set of genes byexperiments designed to find genes whose products changed in response towounding.

Manipulation of one or more wounding responsive gene activities isuseful to modulate the biological activities and/or phenotypes listedbelow. Wounding responsive genes and gene products can act alone or incombination with genes induced in other ways. Useful combinationsinclude wounding responsive genes and/or gene products with similartranscription profiles, similar biological activities, or members offunctionally related biochemical pathways. Whole pathways or segments ofpathways are controlled by transcription factor proteins and proteinscontrolling the activity of signal transduction pathways. Therefore,manipulation of the levels of such proteins is especially useful foraltering phenotypes and biochemical activities of plants. The MA_diffTable(s) reports the transcript levels of the experiment (see EXPT ID:108574, 108575, 108524, 108525, and Wounding (relating to SMD 3714, SMD3715)). For transcripts that had higher levels in the samples than thecontrol, a “+” is shown. A “−” is shown for when transcript levels werereduced in root tips as compared to the control. For more experimentaldetail see the Example section below.

Wounding genes are those sequences that showed differential expressionas compared to controls, namely those sequences identified in theMA_diff tables with a “+” or “−” indication.

Wounding Genes Identified by Cluster Analyses of Differential Expression

Wounding Genes Identified by Correlation to Genes that areDifferentially Expressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of Wounding genes is any group in the MA_clust thatcomprises a cDNA ID that also appears in Expt ID 108574, 108575, 108524,108525, and Wounding (relating to SMD 3714, SMD 3715) of the MA_difftable(s).

Wounding Genes Identified by Correlation to Genes that CausePhysiological Consequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of Woundinggenes. A group in the MA_clust is considered a Wounding pathway ornetwork if the group comprises a cDNA ID that also appears in Knock-inor Knock-out tables that causes one or more of the phenotypes describedin section above.

Wounding Genes Identified by Amino Acid Sequence Similarity

Wounding genes from other plant species typically encode polypeptidesthat share amino acid similarity to the sequences encoded by corn andArabidopsis Wounding genes. Groups of Wounding genes are identified inthe Protein Group table. In this table, any protein group that comprisesa peptide ID that corresponds to a cDNA ID member of a Wounding pathwayor network is a group of proteins that also exhibits Woundingfunctions/utilities.

Such wounding responsive genes and gene products can function either toincrease or dampen the phenotypes and activities below, either inresponse to wounding or in the absence of wounding.

Further, promoters of wounding responsive genes, as described in theReference tables, for example, are useful to modulate transcription thatis induced by wounding or any of the following phenotypes or biologicalactivities below.

III.E.4.a. Use of Wounding-Responsive Genes to Modulate Phenotypes

Wounding responsive genes and gene products can be used to alter ormodulate one or more phenotypes including growth rate; whole plantheight, width, or flowering time; organs (such as coleoptile elongation,young leaves, roots, lateral roots, tuber formation, flowers, fruit, andseeds); biomass; fresh and dry weight during any time in plant life,such as at maturation; number of flowers; number of seedsm seed yield,number, size, weight, harvest index (such as content and composition,e.g., amino acid, nitrogen, oil, protein, and carbohydrate); fruityield, number, size, weight, harvest index, post harvest quality,content and composition (e.g., amino acid, carotenoid, jasmonate,protein, and starch); seed and fruit development; germination of dormantand non-dormant seeds; seed viability, seed reserve mobilization, fruitripening, initiation of the reproductive cycle from a vegetative state,flower development time, insect attraction for fertilization, time tofruit maturity, senescence; fruits, fruit drop; leaves; stress anddisease responses; drought; heat and cold; wounding by any source,including wind, objects, pests and pathogens; uv and high light damage(insect, fungus, virus, worm, nematode damage).

To regulate any of the phenotype(s) above, activities of one or more ofthe wounding responsive genes or gene products can be modulated and theplants can be tested by screening for the desired trait. Specifically,the gene, mRNA levels, or protein levels can be altered in a plantutilizing the procedures described herein and the phenotypes can bescreened for variants as in Winkler et al. (1998) Plant Physiol 118:743-50 and assayed, for example, in accordance with Johnson et. al.(1998) Plant Physiol 116:643-649, Reymond et. al. (2000) Plant Cell 12707-720, or Keith et. al. (1991) Proc. Nat. Acad. Sci. USA 888821 8825.

III.E.4.b. Use of Wounding-Responsive Genes to Modulate BiochemicalActivities

The activities of one or more of the wounding responsive genes can bemodulated to change biochemical or metabolic activities and/or pathwayssuch as those noted below. Such biological activities are documented andcan be measured according to the citations included in the Table below:

BIOLOGICAL OR METABOLIC ACTIVITIES CITATIONS INCLUDING PROCESS AND/ORPATHWAYS ASSAYS Plant Tissue Cell Damage Repair; Cell Flanders (1990) J.Cell Biol. Proliferation Division 110: 1111-1122 Wound Induced SynthesisOf Jasmonic And Reymond, P and Farmer E. E. Pathways Providing SalicylicAcids And The Current Opinion in Plant Defense Against Pathways InducedBy These Biology 1998 1: 404-411 Pests And Pathogens SignalingMolecules. Creelman, RA and Mullet, J. E. Induction Of Jasmonic Acid(1997) Ann Rev. Plant Independent Defense Physiol Mol Biol 48: 355-387Pathways. Leon et al. 1998 Mol Gen Induction Of Lipoxygenase, Genet 254:412-419 Thionins And Nodulins Titarentko et al. 1997 Plant Physiol 115:817-826 Cell Wall Degradation, Rojo, E. et al. 1998. Plant J EthyleneFormation, Systemic 13: 153-165 Signaling And Induction Of Ryan, CA andPearce, G. Defense Related Genes 1998. Ann Rev. Cell Dev. Biol 14: 1-17Specific Rnase Induction Reymond, P. et al. 2000. Plant Cell 12: 707-720Glazebrook, J. 1999. Current Opinion in Plant Biol. 2: 280-286 O'DonnelP. J., et al. 1996 Science 274: 1914-1917 Rojo et al. 1999. Plant J. 20:135-142 Merkouropoulus G. et al. 1999 Planta 208: 212-219 Kariu et al.1998. Bioscience Biotechnology and Biochemistry 62: 1144-1151 Mcoann etal. 1997 PNAS 94: 5473-5477 Other Stress Induced Abscisic Acid FormationAnd Carrera, E and Prat, S. 1998. Pathways Its Signaling Pathway Plant J15: 767-771 Cold Responsive Genes and Chao et. al. 1999. Plant PathwaysPhysiol 120: 979-992 Drought Induced Dehydrins And Pathways ModifiedLipid Membrane Lipid Synthesis Martin, M et al. 1999 Europe MotabolismIncluding Omega-3 Fatty J. Biochem 262: 283-290 Acid Desaturase LipasesLipid Transfer Proteins Modified Sugar And Induction Of GlycohydrolasesEnergy Metabolism And Glycotransferases, Amylases Modified Protein AndInduction Of Nitrogen Metabolism Aminotransferases, Arginase, ProteasesAnd Vegetative Storage Proteins, Aromatic Amino Acid Synthesis SecondaryMetabolite Aromatic Amino Acid Keith, B et al. 1991 PNAS 88: InductionSynthesis And Secondary 8821-8825 Metabolites

Other biological activities that can be modulated by wound responsivegenes and their products are listed in the Reference tables. Assays fordetecting such biological activities are described in the Protein Domaintable.

The MA_diff table reports the changes in transcript levels of variouswound responsive genes in the aerial parts of a plant, 1 and 6 hoursafter the plants were wounded with forceps. The comparison was made withaerial tissues from unwounded plants.

The data from this time course reveal a number of types of woundresponsive genes and gene products, including “early responders,” and“delayed responders.” Profiles of the individual wounding responsivegenes are shown in the Table below together with examples of the kindsof associated biological activities that are modulated when theactivities of one or more such genes vary in plants.

EXAMPLES OF TRANSCRIPT TYPES OF PHYSIOLOGICAL BIOCHEMICAL LEVELS GENESCONSEQUENCES ACTIVITY Up Regulated Early Responders Induction Of KeyTranscription Factors Transcripts To Wounding Signaling Pathways KinasesAnd (Level At 1 h ≈ 6 h) Within And Between Phosphatases Or Cells (LevelAt 1 h > 6 h) Modulation Of Jasmonic Wounding And Stress Acid, SalicylicAcid Induced Signal And Nitric Oxide Transduction Pathways PathwayProteins. Specific Gene Glycohydrolases Transcription InitiationDehydrins Induction Of Repair Rnases Processes Or Cell Death MetabolicEnzymes Nodulins Cell Division And Cell Wall Proteins Reorientation OfCold Response Metabolism, Including Proteins Management Of ActiveLipoxygenase Oxygen Jacalin Proteins To Detoxify Active Oxygen SpeciesMovement Of Wound Systemin Induced Signals Through Plant Synthesis OfBiosynthetic Phytoalexins And Enzymes Secondary Metabolites Up RelatedDelayed Maintenance Of Transcription Factors Transcripts RespondersDefence Pathways Kinases And (Level At 1 h < 6 h) Phosphatases GenesInvolved In Maintenance Of Jasmonic Wounding Reorientated MetabolismAcid, Salicylic Acid Response At And Nitric Oxide Distant Sites FromPathway Proteins Wound. Genes Involved In Maintenance Of WoundGlycohydrolases Maintenance Of Response Dehydrins Wounding ProgrammedCell Death Rnases Response In Selected Cells Metabolic EnzymesReorientation Of Nodulins Metabolism Cold Response Proteins LipoxygenaseJacalin Proteins To Detoxify Active Oxygen Species Cell Division AndCell Wall Proteins Movement Of Wound Systemin Induced Signals ThroughPlant Synthesis Of Biosynthetic Phytoalexins And Enzymes SecondaryMetabolites Down-Regulated Early Negative Regulation Of TranscriptionFactors Transcripts Responder Wounding Response Change In Protein (LevelAt 1 h ≈ 6 h) Repressors Of Pathways Released Structure By Or WoundingPhosphory-Laton (Level At 6 Hr > 1 h) Response (Kinases) Or StateDephos-Phorylation (Phosphatases) Change In Chromatin Structure And OrDna Topology Genes With Changes In Pathways Local Changes InDiscontinued And Processes Operating Regulatory Proteins, Expression OrIn Cells Metabolic Enzymes, UnsTable Transporters Etc. mRNA FollowingWounding Down-Regulated Delayed Negative Regulation Of TranscriptionTranscripts Repressors Of Wounding Response Factors, (Level At 1 hr > 6h) Wounding Pathways Released Phosphatases, Response State KinasesChanges In Protein Complex Structures Chromatin Restructuring ProteinsGenes With Change In Pathways And Local Changes In Discontinued ProcessOperating In Regulatory Proteins, Expression Or Cells Metabolic Enzymes,UnsTable mRNA Transporters Etc. Following Programmed Cell Death MostProteins In Wounding Selected Cells Undergoing Death

Further, any desired sequence can be transcribed in similar temporal,tissue, or environmentally specific patterns as the wounding responsivegenes when the desired sequence is operably linked to a promoter of awounding responsive gene.

III.E.5. Methyl Jasmonate (Jasmonate) Responsive Genes, Gene Componentsand Products

Jasmonic acid and its derivatives, collectively referred to asjasmonates, are naturally occurring derivatives of plant lipids. Thesesubstances are synthesized from linolenic acid in alipoxygenase-dependent biosynthetic pathway. Jasmonates are signallingmolecules which have been shown to be growth regulators as well asregulators of defense and stress responses. As such, jasmonatesrepresent a separate class of plant hormones.

Changes in external or internal jasmonate concentration result inmodulation of the activities of many genes and gene products. Examplesof such “jasmonate responsive” genes and gene products are shown in theReference and Sequence Tables. These genes and/or products areresponsible for effects on traits such as plant vigor and seed yield,especially when plants are growing in the presence of biotic or abioticstresses. They were discovered and characterized from a much larger setof genes by experiments designed to find genes whose mRNA productschanged in concentration in response to application of methyl jasmonateto plants.

Manipulation of one or more jasmonate responsive gene activities isuseful to modulate the biological activities and/or phenotypes testedbelow. Jasmonate response genes and gene products can act alone or incombination. Useful combinations include jasmonate responsive genesand/or gene products with similar transcription profiles, similarbiological activities, or members of the same co-regulated orfunctionally related biochemical pathways. Whole pathways or segments ofpathways are controlled by transcription factor proteins and proteinscontrolling the activity of signal transduction pathways. Therefore,manipulation of such protein levels is especially useful for alteringphenotypes and biochemical activities Such jasmonate responsive genesand gene products can function to either increase or dampen thephenotypes or activities below either in response to changes injasmonate concentration or in the absence of jasmonate fluctuations. TheMA_diff Table(s) reports the transcript levels of the experiment (seeEXPT ID: 108568, 108569, 108555). For transcripts that had higher levelsin the samples than the control, a “+” is shown. A “−” is shown for whentranscript levels were reduced in root tips as compared to the control.For more experimental detail see the Example section below.

MeJA genes are those sequences that showed differential expression ascompared to controls, namely those sequences identified in the MA_difftables with a “+” or “−” indication.

MeJA Genes Identified by Cluster Analyses of Differential Expression

MeJA Genes Identified by Correlation to Genes that are DifferentiallyExpressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of MeJA genes is any group in the MA_clust thatcomprises a cDNA ID that also appears in Expt ID 108568, 108569, 108555of the MA_diff table(s).

MeJA Genes Identified by Correlation to Genes that Cause PhysiologicalConsequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of MeJAgenes. A group in the MA_clust is considered a MeJA pathway or networkif the group comprises a cDNA ID that also appears in Knock-in orKnock-out tables that causes one or more of the phenotypes described insection above.

MeJA Genes Identified by Amino Acid Sequence Similarity

MeJA genes from other plant species typically encode polypeptides thatshare amino acid similarity to the sequences encoded by corn andArabidopsis MeJA genes. Groups of MeJA genes are identified in theProtein Group table. In this table, any protein group that comprises apeptide ID that corresponds to a cDNA ID member of a MeJA pathway ornetwork is a group of proteins that also exhibits MeJAfunctions/utilities.

Further, promoters of jasmonate responsive genes, as described in theReference tables, for example, are useful to modulate transcription thatis induced by jasmonate or any of the following phenotypes or biologicalactivities below.

III.E.5.a. Use of Jasmonate Responsive Genes to Modulate Phenotypes:

Jasmonate responsive genes and their gene products can be used to alteror modulate one or more phenotypes including growth rate, whole plant(including height, flowering time, etc.), seedling, organ, coleoptileelongation, young leaves, roots, lateral roots, tuber formation,flowers, fruit, seeds, biomass; fresh and dry weight during any time inplant life, including maturation and senescence; number of flowers,number of seeds (including secondary metabolite accumulation, alkaloids,anthocyanins; paclitaxel and related taxanes, rosmarinic; seed yield(such as number, size, weight, harvest index, content and composition,e.g., amino acid, jasmonate, oil, protein, and starch); fruit yield(such as number, size, weight, harvest index, post harvest quality,content and composition e.g., amino acid, carotenoid, jasmonate,protein, starch); seed and fruit development; germination of dormant andnon-dormant seeds; seed viability; seed reserve mobilization; fruitripening (such as initiation of the reproductive cycle from a vegetativestate); flower development time; insect attraction for fertilization;time to fruit maturity; senescence; fruits, fruit drop; leaves; stressand disease responses; drought; wounding; UV damage; and insect, fungus,virus, or worm damage.

Further, any desired sequence can be transcribed in similar temporal,tissue, or environmentally specific patterns as the jasmonate responsivegenes when the desired sequence is operably linked to a promoter of ajasmonate responsive gene.

To improve any of the phenotype(s) above, activities of one or more ofthe jasmonate responsive genes or gene products can be modulated and theplants can be tested by screening for the desired trait. Specifically,the gene, mRNA levels, or protein levels can be altered in a plantutilizing the procedures described herein and the phenotypes can beassayed, for example, in accordance to citations described below.

III.E.5.b. Use of Jasmonate-Responsive Genes to Modulate BiochemicalActivities:

The activities of one or more of the jasmonate responsive genes can bemodulated to change biochemical or metabolic activities and/or pathwayssuch as those noted below. Such biological activities are documented andcan be measured according to the citations included in the Table below:

BIOCHEMICAL OR METABOLIC ACTIVITIES AND/OR CITATIONS INCLUDING PROCESSPATHWAYS ASSAYS Turnover of proteins Induction of various This study.Standard proteases, ubiquitin and biochemical assays. proteosomecomponents and turnover of RNA polymerases and translation initiationfactors Reduction in many ribosomal proteins Activation of nitrogenInduction of glutamine Crawford (1995) Plant Cell metabolism synthetase,many 7, 859-868 aminotransferases, This study. Standard vegetativestorage proteins biochemical assays. Lipid turnover Induction of variousThis study. Standard lipases, desaturases, and biochemical assays.reduction of lipid transfer protein mRNAs Sugar metabolism Induction ofsugar This study. Standard transporters, UDP biochemical assays.glucosyltransferases, other transferases Glycolysis and centralInduction of glycolytic This study. Standard carbon metabolism relatedenzymes. Example, biochemical assays. glucose 6-phosphate dehydrogenase,glyceraldehyde-3- phosphate dehydrogenase, phosphoglycerate kinase,phosphoglucomutase ATP synthase Chlorosis Degradation of Tsuchiya et al.(1999) Proc. Chlorophyll Natl. Acad. Sci. USA 96: 15362-15367 Inhibitionof Reinbothe et al. (1993) J. Photosynthesis Related Biol. Chem. 268,10606-10611 Proteins Carbon Assimilation and Induction of chlorophyll abReinbothe et al. (1993) J. turnover binding protein precursor Biol.Chem. 268, 10606-10611 Jasmonate metabolism Induction of lipid Thisstudy. Standard biosynthesis, myrosinase biochemical assays. and jacalinJasmonate mediated signal Receptor binding Cho and Pai (2000) Moltransduction Cells 10, 317-324 Protein kinases Lee et al. (1998) Mol.Gen. Genet. 259, 516-522 Seo et al. (1999) Plant Cell 11, 289-298 Yoonet al. (1999) Plant Mol. Biol. 39, 991-1001 Ubiquitination of Xie et al.(1998) Science 280, Repressor Proteins 1091-1094 Calcium Flux regulatorsBergey and Ryan (1999) Plant Mol. Biol. 40, 815-823 TranscriptionActivators. Xiang et al. (1996) Plant Example-induction of Mol. Biol.32, 415-426 various zinc finger, myb Menke et al. (1999) EMBO J. andAP-2 related factors 18, 4455-4463 Response to Cell Lipid PeroxidationDubery et al. (2000) Mol. Membrane Damage Cell Biol. Res. Commun. 3,105-110 Cell Elongation Inhibition of incorporation Burnett et al.(1993) Plant of Glucose into Cell Wall Physiol. 103, 41-48 SaccharidesCell Organization and Reductions in Ishikawa et al. (1994) PlantDivision tropomyosin related Mol. Biol. 26, 403-414 proteins and certaincyclins Induction of actins and tubulins Cell Wall Turnover andInduction of cell wall Creelman et al. (1992) Proc. modulation proteins,glycine-rich Natl. Acad. Sci. USA 89, proteins, annexins, pectate4938-4941 lyase and pectin esterases Garcia-Muniz et al. (1998)Reductions in various Plant Mol. Biol. 38, 623-632 dehydrins andexpansins Norman et al (1999) Mol. Plant Microbe Interact. 12, 640-644Stress, Disease, and Induction of antifungal Hildmann et al. (1992)Plant Pathogen Resistance proteins, wounding Cell 4, 1157-1170responsive proteins, Reinbothe et al. (1994) Proc. dehydrins, heat shocktype Natl. Acad. Sci. USA 91, proteins and elicitor 7012-7016 responseproteins Moons et al. (1997) Plant Cell 9, 2243-2259 Richard et al.(2000) Plant Mol. Biol. 43, 1-10 Van Wees et al. (2000) Proc. Natl.Acad. Sci. USA 97, 8711-8716 Phytoalexin Biosynthesis Creelman et al.(1992) Proc. Natl. Acad. Sci. USA 89, 4938-4941 Choi et al. (1994) Proc.Natl. Acad. Sci. USA 91, 2329-2333 Biosynthesis of phenolics Doares etal., (1995) Proc. Natl. Acad. Sci. USA 92, 4095-5098 Production ofProtease Botella et al. (1996) Plant Inhibitors Physiol 112, 1201-1210Defense Gene Mason et al. (1993) Plant Transcription in Response Cell 5,241-251 to UV Schaller et al. (2000) Planta 210, 979-984 SecondaryMetabolite Fruit Cartenoid Czapski and Saniewski biosynthesisComposition (1992) J. Plant Physol. 139, 265-268 Palitaxel and RelatedYukimune et al. (1996) Taxanes Nature Biotech. 14, 1129-1132 AlkaloidsAerts et al. (1994) Plant J. 4, 635-643 Geerlings et al. (2000) J. Biol.Chem. 275, 3051-3056 Anthocyanins Franceschi et al. (1991) Proc. Natl.Acad. Sci. USA 83, 6745-6749 Rosmarinic Mizukami et al., (1993) PlantCell Reprod. 12, 706-709 Activation of Ethylene- Czapski and Saniewskiforming Enzyme and (1992) J. Plant Physiol. 139, Production of Ethylene265-268

Other biological activities that can be modulated by the jasmonateresponsive genes and their products are listed in the Reference Tables.Assays for detecting such biological activities are described in theDomain section of the Reference Tables.

Jasmonate responsive genes are characteristically differentiallytranscribed in response to fluctuating jasmonate levels orconcentrations, whether internal or external to an organism or cell. TheMA_diff table(s) report(s) the changes in transcript levels of variousjasmonate responsive genes in the aerial parts of a seedling at 1 and 6hours after being sprayed with Silwet L-77 solution enriched with methyljasmonate as compared to seedlings sprayed with Silwet L-77 alone.

The data from this time course reveal a number of types of jasmonateresponsive genes and gene products, including “early responders” and“delayed responders”. Profiles of the individual kinds of jasmonateresponsive genes are shown in the Table below, together with examples ofthe kinds of associated biological activities that are modulated whenthe activities of such genes vary.

GENE FUNCTIONAL TYPE OF EXAMPLES OF EXPRESSION CATEGORY OF BIOLOGICALBIOCHEMICAL LEVELS GENE ACTIVITY ACTIVITY Upregulated Early Respondersto Binding and Transcription Factors genes Jasmonate Perception ofTransporters (Level at 1 hour ≅ 6 hours). Jasmonate Kinases,Phosphatases, Transduction of Leucine-rich Repeat (Level at 1 hour >Jasmonate signal Proteins (LRRs), GTP- 6 hours) tranduction responsebinding proteins (G- pathways proteins), calcium- Initiation of Specificbinding proteins and Gene Transcription to calcium responsivereorientate proteins metabolism Proteases, lipases, glutamine synthetase(GS), arginase, aminotransferases, glycosyltransferases, sugartransporters, cell wall proteins, methyl transferases, glycolyticenzymes. Upregulated Delayed Jasmonate Maintenance of Enzymes of methylgenes Responders Metabolism under jasmonate-induced (Level at 1 hour <high Jasmonate pathways, including 6 hours) Jasmonate signal dehydrin,phytoalexin, Tranduction Response phenolic, carotenoid, Pathwaysalkaloid and Gene Transcription to anthocyanin Reorientate biosynthesis.Metabolism Transcription factors, Gene Transcription to Transporters,Kinases Maintain Reorientated and phosphatases Metabolism Proteases,Lipases, Reorient Cell Glutaminae Division and Cell Synthetase,Arginase, Development Aminotransferases, Lipid Peroxidases,Glycosyltransferases, Sugar transporters, Cell Wall Proteins, GlycolyticEnzymes, Chlorophyll Binding Proteins Transcription factors, kinases,phosphatases, LRRs, G-proteins Actins, Tubulins, Myosins Cyclins,Cyclin-dependent Kinases (CDPKs) Glycosyl Transferases, Glycosylhydrolases, Expansins, Extensins, O-Methyl Transferases Arabinogalactan-proteins (AGPs), Enzymes of Lipid Biosynthesis, Cutinase Down regulatedEarly responders of Relese of Suppression Transcription Factors,transcripts Jasmonate of Jasmonate Induced Kinases, Phosphatases, (levelat 1 hour = Genes with Pathways LRRs, G-Proteins, 6 hours) discontinuedReorientation of Chromatin (level at 6 hours > expression or metabolismRestructuring proteins, 1 hour) unsTable mRNA Ribosomal proteins,following Jasmonate Translation Factors, uptake Histones, RNApolymerases, Pectin esterase, Lipid transfer proteins Down regulatedGenes with Negative Regulation Transcription factors transcriptsDiscontinued of Jasmonate Induced Kinases, Phosphatases (level at 1hour > expression or Pathways Released. Chromatin 6 hours) UnsTable mRNAReorientation of Restructuring Proteins, Following Jasmonate metabolismLRRs, G-proteins uptake Ribosomal proteins, Translation Factors,Histones RNA Polymerases, Cyclins Pectin esterase, Lipid TransferProteins

Use of Promoters of Jasmonate Responsive Genes

Promoters of Jasmonate responsive genes are useful for transcription ofany desired polynucleotide or plant or non-plant origin. Further, anydesired sequence can be transcribed in a similar temporal, tissue, orenvironmentally specific patterns as the Jasmonate responsive geneswhere the desired sequence is operably linked to a promoter of aJasmonate responsive gene. The protein product of such a polynucleotideis usually synthesized in the same cells, in response to the samestimuli as the protein product of the gene from which the promoter wasderived. Such promoter are also useful to produce antisense mRNAs todown-regulate the product of proteins, or to produce sense mRNAs todown-regulate mRNAs via sense suppression.

III.E.6. Reactive Oxygen Responsive Genes, Gene Components and H2O2Products

Often growth and yield are limited by the ability of a plant to toleratestress conditions, including pathogen attack, wounding, extremetemperatures, and various other factors. To combat such conditions,plant cells deploy a battery of inducible defense responses, includingtriggering an oxidative burst. The burst of reactive oxygenintermediates occurs in time, place and strength to suggest it plays akey role in either pathogen elimination and/or subsequent signaling ofdownstream defense functions. For example, H₂O₂ can play a key role inthe pathogen resistance response, including initiating thehypersensitive response (HR). HR is correlated with the onset ofsystemic acquired resistance (SAR) to secondary infection in distaltissues and organs.

Changes in reactive oxygen, such as H₂O₂ or O₂ ⁻, in the surroundingenvironment or in contact with a plant results in modulation of theactivities of many genes and hence levels of gene products. Examples ofsuch reactive oxygen responsive genes and gene products are shown in theReference, Sequence, Protein Group, Protein Group Matrix, MA_diff andMA_clust tables. These genes and/or products are responsible for effectson traits such as plant vigor and seed yield. The genes were discoveredand characterized from a much larger set by experiments designed to findgenes whose mRNA products changed in response to application of reactiveoxygen, such as H₂O₂, to plants.

Manipulation of one or more reactive oxygen responsive gene activitiesis useful to modulate the following biological activities and/orphenotypes listed below. Reactive oxygen responsive genes and geneproducts can act alone or in combination. Useful combinations includereactive oxygen responsive genes and/or gene products with similartranscription profiles, similar biological activities, or members of thesame or functionally related biochemical pathways. Whole pathways orsegments of pathways are controlled by transcription factor proteins andproteins controlling the activity of signal transduction pathways.Therefore, manipulation of such protein levels is especially useful foraltering phenotypes and biochemical activities of plants.

Such reactive oxygen responsive genes and gene products can function toeither increase or dampen the above phenotypes or activities either inresponse to changes in reactive oxygen concentration or in the absenceof reactive oxygen fluctuations. The MA_diff Table(s) reports thetranscript levels of the experiment (see EXPT ID: 108582, 108583,108537, 108538, 108558, and H2O2 (relating to SMD 7523)). Fortranscripts that had higher levels in the samples than the control, a“+” is shown. A “−” is shown for when transcript levels were reduced inroot tips as compared to the control. For more experimental detail seethe Example section below.

Reactive Oxygen genes are those sequences that showed differentialexpression as compared to controls, namely those sequences identified inthe MA_diff tables with a “+” or “−” indication.

Reactive Oxygen Genes Identified by Cluster Analyses of DifferentialExpression

Reactive Oxygen Genes Identified by Correlation to Genes that areDifferentially Expressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of Reactive Oxygen genes is any group in theMA_clust that comprises a cDNA ID that also appears in Expt ID 108582,108583, 108537, 108538, 108558, and H2O2 (relating to SMD 7523) of theMA_diff table(s).

Reactive Oxygen Genes Identified by Correlation to Genes that CausePhysiological Consequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of ReactiveOxygen genes. A group in the MA_clust is considered a Reactive Oxygenpathway or network if the group comprises a cDNA ID that also appears inKnock-in or Knock-out tables that causes one or more of the phenotypesdescribed in section above.

Reactive Oxygen Genes Identified by Amino Acid Sequence Similarity

Reactive Oxygen genes from other plant species typically encodepolypeptides that share amino acid similarity to the sequences encodedby corn and Arabidopsis Reactive Oxygen genes. Groups of Reactive Oxygengenes are identified in the Protein Group table. In this table, anyprotein group that comprises a peptide ID that corresponds to a cDNA IDmember of a Reactive Oxygen pathway or network is a group of proteinsthat also exhibits Reactive Oxygen functions/utilities.

Further, promoters of reactive oxygen responsive genes, as described inthe Reference tables, for example, are useful to modulate transcriptionthat is induced by reactive oxygen or any of the following phenotypes orbiological activities below.

III.E.6.a. Use of Reactive Oxygen Responsive Genes to ModulatePhenotypes

Reactive oxygen responsive genes and gene products are useful to ormodulate one or more phenotypes including pathogen tolerance and/orresistance; Avr/R locus sensitive; non-host sensitive; HR; SAR (e.g.,where the reactive oxygen responsive gene and products are modulated inconjunction with any of the bacterial, fungal, virus, or other organismlisted below); bacteria resistance, e.g. to Erwinia stewartii,Pseudomonas syringae, Pseudomonas tabaci, Stuart's wilt, etc.; fungalresistance including to downy mildews such as Scleropthora macrospora,Sclerophthora rayissiae, Sclerospora graminicola, Peronosclerosporasorghi, Peronosclerospora philippinensis, Peronosclerospora sacchari,Peronosclerospora maydis; rusts such as Puccinia sorphi, Pucciniapolysora, Physopella zeae, etc.; other fungal diseases such asCercospora zeae-maydis, Colletotrichum graminicola, Fusariummonoliforme, Exserohilum turcicum, Bipolaris maydis, Phytophthoraparasitica, Peronospora tabacina, Septoria, etc.; virus or viroidresistance, e.g. to tobacco or cucumber mosaic virus, ringspot virus,necrosis virus, pelargonium leaf curl virus, red clover mottle virus,tomato bushy stunt virus, and like viruses; insect resistance, such asto aphids e.g. Myzus persicae; beetles, beetle larvae; etc.; nematodes,e.g. Meloidogyne incognita; lepidoptera, e.g. Heliothus spp. etc.;resistance specifically in primary or secondary leaves; stresstolerance; winter survival; cold tolerance; heavy metal tolerance, suchas cadmium; physical wounding; increased organelle tolerance to redoxstress, such as in mitochondria, and chloroplasts; cell death;apoptosis, including death of diseased tissue; senescence; fruit drop;biomass; fresh and dry weight during any time in plant life, such asmaturation; number of flowers, seeds, branches, and/or leaves; seedyield, including number, size, weight, and/or harvest index; fruityield, including number, size, weight, and/or harvest index; plantdevelopment; time to fruit maturity; cell wall strengthening andreinforcement; plant product quality, e.g. paper making quality); foodadditives; treatment of indications modulated by free radicals, andcancer

To regulate any of the phenotype(s) above, activities of one or more ofthe reactive oxygen responsive genes or gene products can be modulatedand the plants can be tested by screening for the desired trait.Specifically, the gene, mRNA levels, or protein levels can be altered ina plant utilizing the procedures described herein and the phenotypes canbe screened for variants as in Winkler et al. (1998) Plant Physiol 118:743-50 and assayed, for example, in accordance to Alvarez et al., (1998)Cell 92: 773-784; Halhbrock and Scheel, (1989) Ann. Rev. Plant Physiol.Plant Mol. Biol. 40: 347-369; Lamb et al., (1997) Ann. Rev. Plant Mol.Biol. Plant Physio. 48: 251-275; Lapwood et al. (1984) Plant Pathol. 33:13-20; Levine et al. (1996) Curr. Biol. 6: 427-437; McKersie et al.,(2000) Plant Physiol. 122(4): 1427-1437; Olson and Varner (1993) PlantJ. 4: 887-892; Pastore et al., (2000), FEBS Lett 470(1): 88-92; Pastoriet al., (1997) Plant Physiol. 113: 411-418. Romero-Puertas et al.,(1999) Free Radic. Res. 1999 31 Suppl: S25-31; Shirataki et al.,Anticancer Res 20(1A): 423-426 (2000); Wu et al., (1995) Plant Cell 7:1357-1368;

III.E.6.b. Use of Reactive Oxygen Responsive Genes to ModulateBiochemical Activities

The activities of one or more of the reactive oxygen responsive genescan be modulated to change biochemical or metabolic activities and/orpathways such as those noted below. Such biological activities aredocumented and can be measured according to the citations above andincluded in the Table below:

BIOCHEMICAL OR METABOLIC ACTIVITIES CITATIONS INCLUDING PROCESS AND/ORPATHWAYS ASSAYS Reinforcement of Modulation Of The Production Of Bradleyet al. 1992. Cell 70, Cell Walls ExtracTable Proline-Rich Protein 21-30Modulation Of Lignification Mansouri et al. (1999) Physiol Plant 106:355-362 Stress, Disease, Induction Of Pathogenesis Related Chamnongpolet. al. (1998) Pathogen Resistance Proteins, Phytoalexins And Many Proc.Nat. Acad Sci USA and Wounding Defense Pathways. 12; 95: 5818-23.Induction Of Detoxifying Davis et al. (1993) Enzymes Such As GlutathioneS- Phytochemistry 32: 607-611. Transferase And Ascorbate Chen et. al.Plant J. (1996) Peroxidase 10: 955-966 Disease Resistance Gadea et. al.(1999) Mol Gen Genet 262: 212-219 Wu et. al. (1995) Plant Cell 7:1357-68 Reactive Oxygen Generation Orozco-Cardenas and Ryan FollowingWounding And (1999) Proc. Nat. Acad. Sci. Changes In Physical PressureUSA 25; 96: 6553-7. Yahraus et al. (1995) Plant Physiol. 109: 1259-1266Modulation Of Genes Involved In LEGENDRE ET AL. (1993) Wound Repair AndCell Division PLANT PHYSIOL. 102: 233-240 Modulation Of Nitric OxideDELLEDONNE ET AL. Signaling (1998) NATURE 394: 585-588 Salicyclic AcidAccumulation And DURNER AND KLESSIG Signaling (1996) J. BIOL. CHEM. 271:28492-501 Programmed Cell Induction Of Cell Death Pathway LEVINE ET AL.(1996) Death Genes CURR. BIOL. 6: 427-437. REYNOLDS ET. AL. (1998)BIOCHEM. J. 330: 115-20

Other biological activities that can be modulated by the reactive oxygenresponsive genes and their products are listed in the Reference tables.Assays for detecting such biological activities are described in theProtein Domain table.

Reactive oxygen responsive genes are characteristically differentiallytranscribed in response to fluctuating reactive oxygen levels orconcentrations, whether internal or external to an organism or cell. TheMA_diff table reports the changes in transcript levels of variousreactive oxygen responsive genes in the aerial parts of a plant at 1 and6 hours after the plant was sprayed with Silwett L-77 solution enrichedwith hydrogen peroxide as compared to plants sprayed with Silwett L-77alone.

The data from this time course reveal a number of types of reactiveoxygen responsive genes and gene products, including “early responders,”and “delayed responders”. Profiles of individual reactive oxygenresponsive genes are shown in the Table below together with examples ofwhich associated biological activities are modulated when the activitiesof one or more such genes vary in plants.

EXAMPLES OF GENE FUNCTIONAL BIOCHEMICAL EXPRESSION CATEGORY OFPHYSIOLOGICAL ACTIVITY LEVELS GENE CONSequence OF GENE PRODUCTSUpregulated Early Responders Perceiving Transcription Factorstranscripts To Reactive Oxygen Kinases And Phosphatases (Higher at 1 hReactive Oxygen Reactive Oxygen Transporters Than 6 h) ResponseGlutathione S-Transferase (Level at 1 h ≅ Transduction Heat ShockProteins 6 h) Pathways Salicylic Acid Response Initiating SpecificPathway Proteins Gene Transcription Jasmonic Acid Pathway ProteinsDehydrins Peroxidases Catalase Proteases Pathogen Response Proteins Ca2+ Channel Blockers Phenylalanine Ammonia Lyase Upregulated DelayedReactive Maintenance Of Transcription Factors transcripts Oxygen DefencePathways Kinases And Phosphatases (Lower at 1 h Responders To ControlActive Reactive Oxygen Than 6 h) Oxygen Scavenging Enzymes Activation OfCell Cell Wall And Cell Death Pathways In Division/Growth PromotingSpecific Cells Pathway Enzymes Pathogen Response Proteins Proteins OfDefence Pathways Proteases, Cellulases, Nucleases And Other DegradingEnzymes. Membrane Proteins Mitochondrial And Chloroplast Energy RelatedProteins Downregulated Early Responder Negative Transcription Factorstranscripts Repressors Of Regulation Of Kinases And Phosphatases Levelat 1 h ≅ 6 h Reactive Oxygen Reactive Oxygen- Chromatin RemodellingLevel at 6 h > 1 h. Response Inducible Pathways Proteins Down RegulatedPathways Released Metabolic Enzymes In Transcripts Genes Of Reduction InAffected Cells (Level at 1 h > 6 h Pathways That Activities Of MembraneProteins And Are Minimized In Pathways Not Cell Wall Proteins ResponseTo Maintained Under Transcription Factors Reactive Oxygen High ReactiveKinases And Phosphatases Delayed Oxygen Chromatin Remodelling ResponderNegative Proteins Repressors Of Regulation Of Metabolic Enzymes InReactive Oxygen Reactive Oxygen Affected Cells Response InduciblePathways Membrane Proteins And Pathways Released Cell Wall ProteinsGenes Of Reduction In Many Proteins In Cells Pathways That Activities OfUndergoing Cell Death Or In Are Minimised In Pathways Not Damaged CellsResponse To Maintained Under Reactive Oxygen Reactive Oxygen ProgrammedCell Death

Further, promoters of reactive oxygen responsive genes, as described inthe Reference tables, for example, are useful to modulate transcriptionthat is induced by reactive oxygen or any of the following phenotypes orbiological activities below.

III.E.7. Salicylic Acid Responsive Genes, Gene Components and Products

Plant defense responses can be divided into two groups: constitutive andinduced. Salicylic acid (SA) is a signaling molecule necessary foractivation of the plant induced defense system known as systemicacquired resistance or SAR. This response, which is triggered by priorexposure to avirulent pathogens, is long lasting and provides protectionagainst a broad spectrum of pathogens. Another induced defense system isthe hypersensitive response (HR). HR is far more rapid, occurs at thesites of pathogen (avirulent pathogens) entry and precedes SAR. SA isalso the key signaling molecule for this defense pathway.

Changes in SA concentration in the surrounding environment or within aplant results in modulation of many genes and gene products. Examples ofsuch SA responsive genes and gene products are shown in the Reference,Sequence, Protein Group, Protein Group Matrix, MA_diff and MA_clusttables. These genes and/or products are responsible for effects ontraits such as plant vigor and seed yield. They were discovered andcharacterized from a much larger set by experiments designed to findgenes whose mRNA products changed in response to SA treatment.

While SA responsive polynucleotides and gene products can act alone,combinations of these polynucleotides also affect growth anddevelopment. Useful combinations include different SA responsivepolynucleotides and/or gene products that have similar transcriptionprofiles or similar biological activities, and members of the same orsimilar biochemical pathways. In addition, the combination of SAresponsive polynucleotides and/or gene product with anotherenvironmentally responsive polynucleotide is also useful because of theinteractions that exist between hormone-regulated pathways, stress andpathogen induced pathways, nutritional pathways and development. Here,in addition to polynucleotides having similar transcription profilesand/or biological activities, useful combinations includepolynucleotides that may have different transcription profiles but whichparticipate in common and overlapping pathways.

Such SA responsive genes and gene products can function to eitherincrease or dampen the above phenotypes or activities either in responseto changes in SA concentration or in the absence of SA fluctuations. TheMA_diff Table(s) reports the transcript levels of the experiment (seeEXPT ID: 108586, 108587, 108515, 108552, 108471, 108472, 108469, 108470,107953, 107960, 108443, 108440, 108441, 108475, 108476). For transcriptsthat had higher levels in the samples than the control, a “+” is shown.A “−” is shown for when transcript levels were reduced in root tips ascompared to the control. For more experimental detail see the Examplesection below.

SA genes are those sequences that showed differential expression ascompared to controls, namely those sequences identified in the MA_difftables with a “+” or “−” indication.

SA Genes Identified by Cluster Analyses of Differential Expression

SA Genes Identified by Correlation to Genes that are DifferentiallyExpressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of SA genes is any group in the MA_clust thatcomprises a cDNA ID that also appears in Expt ID 108586, 108587, 108515,108552, 108471, 108472, 108469, 108470, 107953, 107960, 108443, 108440,108441, 108475, 108476 of the MA_diff table(s).

SA Genes Identified by Correlation to Genes that Cause PhysiologicalConsequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of SA genes.A group in the MA_clust is considered a SA pathway or network if thegroup comprises a cDNA ID that also appears in Knock-in or Knock-outtables that causes one or more of the phenotypes described in sectionabove.

SA Genes Identified by Amino Acid Sequence Similarity

SA genes from other plant species typically encode polypeptides thatshare amino acid similarity to the sequences encoded by corn andArabidopsis SA genes. Groups of SA genes are identified in the ProteinGroup table. In this table, any protein group that comprises a peptideID that corresponds to a cDNA ID member of a SA pathway or network is agroup of proteins that also exhibits SA functions/utilities.

Further, promoters of SA responsive genes, as described in the Referencetables, for example, are useful to modulate transcription that isinduced by SA or any of the following phenotypes or biologicalactivities below.

III.E.7.a. Use of Salicylic Acid-Responsive Genes to Modulate Phenotypes

SA responsive genes and gene products are useful to or modulate one ormore phenotypes including pathogen tolerance and/or resistance; Avr/Rlocus Interactions; non-host interactions; HR; SAR, e.g., SA responsivegenes and/or products in conjunction with any of the organisms listedbelow; resistance to bacteria e.g. to Erwinia stewartii, Pseudomonassyringae, Pseudomonas tabaci, Stuart's wilt, etc.; resistance to fungie.g. to Downy mildews such as Scleropthora macrospora, Sclerophthorarayissiae, Sclerospora graminicola, Peronosclerospora sorghi,Peronosclerospora philippinensis, Peronosclerospora sacchari,Peronosclerospora maydis; rusts such As Puccinia sorphi, Pucciniapolysora, Physopella zeae, etc.; and to other fungal diseases e.g.Cercospora zeae-maydis, Colletotrichum graminicola, Fusariummonoliforme, Exserohilum turcicum, Bipolaris maydis, Phytophthoraparasitica, Peronospora tabacina, Septoria, etc.; resistance to virusesor viroids e.g., to Tobacco or Cucumber Mosaic Virus, Ringspot Virus,Necrosis Virus, Pelargonium Leaf Curl Virus, Red Clover Mottle Virus,Tomato Bushy Stunt Virus, and like viruses; resistance to insects, suchas to aphids e.g. Myzus persicae; to beetles and beetle larvae; tolepidoptera larvae e.g. Heliothus etc.; resistance to nematodes, e.g.Meloidogyne incognita etc.; local resistance in primary (infected) orsecondary (uninfected) leaves; stress tolerance; winter survival; coldtolerance; salt tolerance; heavy metal tolerance, such as cadmium;tolerance to physical wounding; increased organelle tolerance to redoxstress (such as in mitochondria, and chloroplasts); cell death;programmed cell death, including death of diseased tissue and duringsenescence); fruit drop; biomass; fresh and dry weight during any timein plant life, such as maturation; number of flowers, seeds, branches,and/or leaves; seed yield, including number, size, weight, and/orharvest index; fruit yield, including number, size, weight, and/orharvest index; plant development; time to fruit maturity; cell wallstrengthening and reinforcement; plant product quality; e.g. papermaking quality);food additives; treatment of indications modulated byfree radicals; and cancer.

To regulate any of the desired phenotype(s) above, activities of one ormore of the SA responsive genes or gene products can be modulated andthe plants tested by screening for the desired trait. Specifically, thegene, mRNA levels, or protein levels can be altered in a plant utilizingthe procedures described herein and the phenotypes can be assayed. As anexample, a plant can be transformed according to Bechtold and Pelletier(1998, Methods. Mol. Biol. 82:259-266) and/or screened for variants asin Winkler et al. (1998) Plant Physiol 118: 743-50 and visuallyinspected for the desired phenotype or metabolically and/or functionallyassayed according to Zhao et al. (1998, Plant Cell 10:359-70) andAlvarez et al. (1998, Cell 92: 733-84).

III.E.7.b. Use of Salicylic Acid-Responsive Genes to ModulateBiochemical Activities

The activities of one or more of the SA responsive genes can bemodulated to change biochemical or metabolic activities and/or pathwayssuch as those noted below. Such biological activities can be measuredaccording to the citations included in the Table below:

BIOCHEMICAL OR METABOLIC ACTIVITIES CITATION INCLUDING PROCESS AND/ORPATHWAYS ASSAYS Protection Systemic Acquired Resistance Alvarez et al.(1998) Cell From (SAR) 92: 733-84 Microbial Phytoalexin BiosynthesisLapwood et al. (1984) Plant Pathogens PR Protein Biosynthesis Pathol.33: 13-20 Local Resistance Davis et al. (1993) Wound ResponsePhytochemistry 32: 607-11 Yahraus et al. (1995) Plant Physiol. 109:1259-66 Cell Modulation Of Reactive Alvarez et al. (1998) Cell SignalingOxygen Signaling 92: 773-784 Modulation Of No Signaling Delledonne etal. (1998) Nature 394: 585-588 Growth Lignification Redman et al. (1999)Plant And Physiol. 119: 795-804 Development

Other biological activities that can be modulated by the SA responsivegenes and gene products are listed in the Reference tables. Assays fordetecting such biological activities are described in the Protein Domaintable.

Salicylic acid responsive genes are characteristically differentiallytranscribed in response to fluctuating SA levels or concentrations,whether internal or external to an organism or cell. The MA_diff tablereports the changes in transcript levels of various SA responsive genesin entire seedlings at 1 and 6 hours after the seedling was sprayed witha Hoagland's solution enriched with SA as compared to seedlings sprayedwith Hoagland's solution only.

The data from this time course can be used to identify a number of typesof SA responsive genes and gene products, including “early responders”and “delayed responders.” Profiles of these different SA responsivegenes are shown in the Table below together with examples of the kindsof associated biological activities.

EXAMPLES OF GENE FUNCTIONAL BIOCHEMICAL EXPRESSION CATEGORYPHYSIOLOGICAL ACTIVITIES OF GENE LEVELS OF GENE CONSEQUENCES PRODUCTSUpregulated Genes Early SA Perception Transcription Factors (Level At 1h ≅ 6 h) Responders To SA Uptake Transporters, Kinases, Or SA ModulationOf SA Phosphatases, G- (Level At 1 h > 6 h) Response TransductionProteins, LRR, DNA Pathways Remodelling Proteins Upregulated GenesDelayed Specific Defensegene Proteases, PRProteins, (Level At 1 h < 6 h)Responders To Transcription Initiation Cellulases, Chitinases, SA (E. G.Pr Genes, Pal Cutinases, Other Degrading Enzymes, Pal, Proteins OfDefense Pathways, Cell Wall Proteins Epoxide Hydrolases, MethylTransferases Downregulated Early Negative Regulation Transcriptionfactors, (Level At 1 h ≅ 6 h) Responder Of SA Inducible kinases,phosphatases, G- Or Repressors To Pathways Released proteins, LRR,(Level At 6 h > 1 h) SA transporters, calcium Genes With bindingproteins, Discontinued chromatin remodelling Expression Or proteinUnsTable mRNA In The Presence Of SA Down-Regulated Delayed NegativeRegulation Of Transcription Factors, Transcripts Responders To SAInducible Pathways Kinases, Phosphatases, (Level At 1 h > 6 h) SAMetabolism Released G-Proteins, LRR, Genes With Transporters, CalciumDiscontinued Binding Proteins, Expression Or Chromatin RemodellingUnsTable Protein mRNA In The Presence Of SA

Further, any desired sequence can be transcribed in similar temporal,tissue, or environmentally specific patterns as the SA responsive geneswhen the desired sequence is operably linked to a promoter of a SAresponsive gene.

III.E.8. Nitric Oxide Responsive Genes, Gene Components and Products

The rate-limiting element in plant growth and yield is often its abilityto tolerate suboptimal or stress conditions, including pathogen attackconditions, wounding and the presence of various other factors. Tocombat such conditions, plant cells deploy a battery of inducibledefense responses, including synergistic interactions between nitricoxide (NO), reactive oxygen intermediates (ROS), and salicylic acid(SA). NO has been shown to play a critical role in the activation ofinnate immune and inflammatory responses in animals. At least part ofthis mammalian signaling pathway is present in plants, where NO is knownto potentiate the hypersensitive response (HR). In addition, NO is astimulator molecule in plant photomorphogenesis.

Changes in nitric oxide concentration in the internal or surroundingenvironment, or in contact with a plant, results in modulation of manygenes and gene products. Examples of such nitric oxide responsive genesand gene products are shown in the Reference and Sequence Tables. Thesegenes and/or products are responsible for effects on traits such asplant vigor and seed yield. They were discovered and characterized froma much larger set by experiments designed to find genes whose mRNAproducts changed in response to nitric oxide treatment.

While nitric oxide responsive polynucleotides and gene products can actalone, combinations of these polynucleotides also affect growth anddevelopment. Useful combinations include different nitric oxideresponsive polynucleotides and/or gene products that have similartranscription profiles or similar biological activities, and members ofthe same or similar biochemical pathways. Whole pathways or segments ofpathways are controlled by transcription factor proteins and proteinscontrolling the activity of signal transduction pathways. Therefore,manipulation of the levels of such proteins is especially useful foraltering phenotypes and biochemical activities of plants. In addition,the combination of a nitric oxide responsive polynucleotide and/or geneproduct with other environmentally responsive polynucleotides is alsouseful because of the interactions that exist between hormone-regulatedpathways, stress pathways, pathogen stimulated pathways, nutritionalpathways and development. Here, in addition to polynucleotides havingsimilar transcription profiles and/or biological activities, usefulcombinations include polynucleotides that may have differenttranscription profiles but which participate in common or overlappingpathways. The MA_diff Table(s) reports the transcript levels of theexperiment (see EXPT ID: 108584, 108585, 108526, 108527, 108559). Fortranscripts that had higher levels in the samples than the control, a“+” is shown. A “−” is shown for when transcript levels were reduced inroot tips as compared to the control. For more experimental detail seethe Example section below.

NO genes are those sequences that showed differential expression ascompared to controls, namely those sequences identified in the MA_difftables with a “+” or “−” indication.

NO Genes Identified by Cluster Analyses of Differential Expression

NO Genes Identified by Correlation to Genes that are DifferentiallyExpressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of NO genes is any group in the MA_clust thatcomprises a cDNA ID that also appears in Expt ID 108584, 108585, 108526,108527, 108559 of the MA_diff table(s).

NO Genes Identified by Correlation to Genes that Cause PhysiologicalConsequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of NO genes.A group in the MA_clust is considered a NO pathway or network if thegroup comprises a cDNA ID that also appears in Knock-in or Knock-outtables that causes one or more of the phenotypes described in sectionabove.

NO Genes Identified by Amino Acid Sequence Similarity

NO genes from other plant species typically encode polypeptides thatshare amino acid similarity to the sequences encoded by corn andArabidopsis NO genes. Groups of NO genes are identified in the ProteinGroup table. In this table, any protein group that comprises a peptideID that corresponds to a cDNA ID member of a NO pathway or network is agroup of proteins that also exhibits NO functions/utilities.

Such nitric oxide responsive genes and gene products can function eitherto increase or dampen the above phenotypes or activities either inresponse to changes in nitric oxide concentration or in the absence ofnitric oxide fluctuations. Further, promoters of nitric oxide responsivegenes, as described in the Reference tables, for example, are useful tomodulate transcription that is induced by nitric oxide or any of thefollowing phenotypes or biological activities below.

III.E.8.a. Use of Nitric Oxide-Responsive Genes to Modulate Phenotypes:

Nitric oxide responsive genes and gene products are useful to ormodulate one or more phenotypes including Stress Responses, Mediation ofresponse to stresses, Disease resistance, Growth, Roots, Stems, Leaves,Cells, Promotes leaf cell elongation, Biomass; Fresh and Dry Weightduring any time in plant life, such as at maturation; Size and/orWeight; Flowers, Seeds, Branches, Leaves, Roots, Development, SeedDevelopment, Dormancy; Control rate and timing of germination, Prolongsseed storage and viability; and Senescence.

Further, any desired sequence can be transcribed in similar temporal,tissue, or environmentally specific patterns as the nitric responsivegenes when the desired sequence is operably linked to a promoter of anitric responsive gene.

To regulate any of the desired phenotype(s) above, activities of one ormore of the nitric oxide responsive genes or gene products can bemodulated and the plants tested by screening for the desired trait.Specifically, the gene, mRNA levels, or protein levels can be altered ina plant utilizing the procedures described herein and the phenotypes canbe assayed. As an example, a plant can be transformed according toBechtold and Pelletier (1998) Methods. Mol. Biol. 82: 259-266 and/orscreened for variants as described in Winkler et al. (1998) PlantPhysiol. 118: 743-50 and visually inspected for the desired phenotype.Alternatively, plants can be metabolically and/or functionally assayedaccording to Beligni and Lamattina (2000) Planta 210: 215-21), Lapwoodet al (1984) Plant Pathol 33: 13-20, and/or Brown and Botstein (1999)Nature Genet. 21: 33-37.

III.E.8.b. Use of Nitric Oxide-Responsive Genes to Modulate BiochemicalActivities:

The activities of one or more of the nitric oxide responsive genes canbe modulated to change biochemical or metabolic activities and/orpathways such as those noted below. Such biological activities can bemeasured according to the citations included in the Table below:

BIOCHEMICAL OR METABOLIC ACTIVITIES CITATIONS INCLUDING PROCESS AND/ORPATHWAYS ASSAYS Stress Response Programmed Cell Death Levine et al(1996) Curr. Biol 6: 427-37 Sellins and Cohen (1991) Reactive Oxygenbased Defence Radiat. Res. 126: 88-95 Pathways Kumar and Klessig (2000)Mol. Plant Microbe Interact. 13: 347-351 Disease Resistance MicrobialPathogen resistance Lapwood et al (1984) Plant pathways Pathol 33: 13-20Kumar and Klessig (2000) Mol. Plant microbe interact.13: 347-351 Klessiget. al.(2000) Proc. Nat. Acad. Sci USA 97: 8849-8855 Delledonna etal(1998) Nature 394: 585-588 Programmed Cell Death Levine et al (1996)Curr. Biol 6: 427-437 Sellins and Cohen (1991) Radiat. Res. 126: 88-95Cellular Protectant Gene Brown and Botstein (1999) expression Nat Genet21: 33-37 Phytoalexin Biosynthesis Davis et al. (1993) Phytochemistry32: 607-611 Signal Transduction Regulation of hydrogen peroxide Wu etal. (1995) Plant Cell signaling 7, 1357-1368 Reorientation of nitrogenInduction of ribosomal proteins, This study. Standard metabolismasparagine synthesis, proteases, assays for detection of Rnases changesReorientation of sugar and Induction of sugar transporters, This study.Standard energy metabolism ATPases, glycohydrolases, and assays fordetection of glycolytic enzymes, for example changes

Other biological activities that can be modulated by the NO responsivegenes and gene products are listed in the Reference Tables. Assays fordetecting such biological activities are described in the Protein Domaintable.

NO responsive genes are characteristically differentially transcribed inresponse to fluctuating NO levels or concentrations, whether internal orexternal to an organism or cell. The MA_diff table(s) report(s) thechanges in transcript levels of various NO responsive genes in aerialtissues at 1 and 6 hours after a plant was sprayed with a Silwett L-77solution enriched with 5 mM sodium nitroprusside, which is an NO donor.These changes are in comparison with plants sprayed with Silwett L-77solution only.

The data from this time course can be used to identify a number of typesof NO responsive genes and gene products, including “early responders”and “delayed responders” Profiles of these different nitric oxideresponsive genes are shown in the Table below together with examples ofthe kinds of associated biological activities.

GENE FUNCTIONAL EXAMPLES OF EXPRESSION CATEGORY OF PHYSIOLOGICALBIOCHEMICAL LEVEL GENE CONSEQUENCES ACTIVITY Upregulated genes Earlyresponder NO Perception Transcription Factors (level at 1 hour ≅ 6repressors to NO NO Uptake hours) Modulation of NO Transporters (levelat 1 hour > 6 Response Transduction Pathogen responsive hours) Pathwaysproteins, salicylic and jasmonate pathway proteins Specific GeneProteins to provide Transcription Initiation defence against active ofPathways to oxygen e.g. glutathione Optimize NO Response transferase,ascorbate Pathways free radical reductase, ascorbate peroxidase,nitrilase, heat shock proteins Proteins to reorient metabolism e.g.proteases, Rnases, proteasomes, asparagine synthetase, glycohydrolases,transporters Proteins to inhibit transport of nitric oxide Degradationenzymes Upregulated Delayed NO Maintenance of NO Metabolic transcriptsresponders metabolism in presence Pathway enzymes (level at 1 hour < 6of High NO Pathogen responsive hours) Maintenace of disease proteins,salicylic and defence pathways jasmonate pathway proteins Maintenance ofProteins to provide pathways against defence against active reactiveoxygen oxygen e.g. glutathione production transferase, ascorbate freeradical reductase, ascorbate peroxidase, nitrilase, heat shock proteinsMaintenance of Proteins to reorient different metabolic and sustainmetabolism programs e.g. proteases, Rnases, proteasomes, asparaginesynthetase, glycohydrolases, transporters, Proteins to inhibit transportof NO Selective cell death Degradation enzymes Down Regulated Earlyresponders of Negative regulation of Transcription factors TranscriptsNO utilization NO utilization Kinases and (level at 1 hours ≅ 6 pathwayspathways released phosphatases hours) Chromatin (level at 6 hours > 1restructuring proteins hour) Genes with Reorientation of Transcriptiondiscontinued metabolism factors, metabolic expression or enzymes,kinases and unsTable mRNA phosphatases, following nitric oxidetransporters, ribosomal uptake proteins Programmed cell death Mostproteins in cells undergoing cell death Down Regulated Delayed responderNegative regulation of Transcription factors Transcripts repressors ofNO NO utilization Kinases and (level at 1 hour > 6 stress metabolismpathways released phosphatases hours) Chromatin restructuring proteinsGenes with Reorientation of Transcription discontinued metabolismfactors, metabolic expression or enzymes, kinases and unsTablephosphatases, mRNA following transporters, ribosomal nitric oxide uptakeproteins. Programmed cell death Most proteins in cells undergoingprogrammed cell death

Use of Promoters of NO Responsive Genes

Promoters of NO responsive genes are useful for transcription of anydesired polynucleotide or plant or non-plant origin. Further, anydesired sequence can be transcribed in a similar temporal, tissue, orenvironmentally specific patterns as the NO responsive genes where thedesired sequence is operably linked to a promoter of a NO responsivegene. The protein product of such a polynucleotide is usuallysynthesized in the same cells, in response to the same stimuli as theprotein product of the gene from which the promoter was derived. Suchpromoter are also useful to produce antisense mRNAs to down-regulate theproduct of proteins, or to produce sense mRNAs to down-regulate mRNAsvia sense suppression.

III.9. Osmotic Stress Responsive Genes, Gene Components and Products

The ability to endure and recover from osmotic and salt related stressis a major determinant of the geographical distribution and productivityof agricultural crops. Osmotic stress is a major component of stressimposed by saline soil and water deficit. Decreases in yield and cropfailure frequently occur as a result of aberrant or transientenvironmental stress conditions even in areas considered suitable forthe cultivation of a given species or cultivar. Only modest increases inthe osmotic and salt tolerance of a crop species would have a dramaticimpact on agricultural productivity. The development of genotypes withincreased osmotic tolerance would provide a more reliable means tominimize crop losses and diminish the use of energy-costly practices tomodify the soil environment.

Changes in the osmotic concentration of the surrounding environment orwithin a plant results in modulation of many genes and gene products.Examples of such osmotic stress responsive genes and gene products,including salt responsive genes, are shown in the Reference, Sequence,Protein Group, Protein Group Matrix, MA_diff and MA_clust tables. Thesegenes and/or products are responsible for effects on traits such asplant vigor and seed yield.

While osmotic and/or salt stress responsive polynucleotides and geneproducts can act alone, combinations of these polynucleotides alsoaffect growth and development. Useful combinations include differentosmotic stress responsive polynucleotides and/or gene products that havesimilar transcription profiles or similar biological activities, andmembers of the same or similar biochemical pathways. In addition, thecombination of an osmotic stress responsive polynucleotide and/or geneproduct with another environmentally responsive polynucleotide is alsouseful because of the interactions that exist between hormone-regulatedpathways, stress pathways, nutritional pathways and development. Here,in addition to polynucleotides having similar transcription profilesand/or biological activities, useful combinations includepolynucleotides that may have different transcription profiles but whichparticipate in a common pathway.

Such osmotic and/or salt stress responsive genes and gene products canfunction to either increase or dampen the above phenotypes or activitieseither in response to changes in osmotic concentration or in the absenceof osmotic fluctuations. The MA_diff Table(s) reports the transcriptlevels of the experiment (see EXPT ID: 108570, 108571, 108541, 108542,108553, 108539, 108540). For transcripts that had higher levels in thesamples than the control, a “+” is shown. A “−” is shown for whentranscript levels were reduced in root tips as compared to the control.For more experimental detail see the Example section below.

Osmotic Stress genes are those sequences that showed differentialexpression as compared to controls, namely those sequences identified inthe MA_diff tables with a “+” or “−” indication.

Osmotic Stress Genes Identified by Cluster Analyses of DifferentialExpression

Osmotic Stress Genes Identified by Correlation to Genes that areDifferentially Expressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of Osmotic Stress genes is any group in theMA_clust that comprises a cDNA ID that also appears in Expt ID 108570,108571, 108541, 108542, 108553, 108539, 108540 of the MA_diff table(s).

Osmotic Stress Genes Identified by Correlation to Genes that CausePhysiological Consequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of OsmoticStress genes. A group in the MA_clust is considered a Osmotic Stresspathway or network if the group comprises a cDNA ID that also appears inKnock-in or Knock-out tables that causes one or more of the phenotypesdescribed in section above.

Osmotic Stress Genes Identified by Amino Acid Sequence Similarity

Osmotic Stress genes from other plant species typically encodepolypeptides that share amino acid similarity to the sequences encodedby corn and Arabidopsis Osmotic Stress genes. Groups of Osmotic Stressgenes are identified in the Protein Group table. In this table, anyprotein group that comprises a peptide ID that corresponds to a cDNA IDmember of a Osmotic Stress pathway or network is a group of proteinsthat also exhibits Osmotic Stress functions/utilities.

Further, promoters of osmotic stress responsive genes, as described inthe Reference tables, for example, are useful to modulate transcriptionthat is induced by osmotic stress or any of the following phenotypes orbiological activities below.

III.E.9.a. Use of Osmotic Stress Responsive Genes to Modulate Phenotypes

Osmotic stress responsive genes and gene products are useful to ormodulate one or more phenotypes including growth; roots; stems; leaves;development (such as cell growth by DNA synthesis and cell division,seed development (with regard to desiccation tolerance and dormancy,such as control rate of germination and prolongs seed storage andviability and senescence); stress responses; desiccation; drought; andsalt.

To regulate any of the phenotype(s) above, activities of one or more ofthe osmotic stress responsive genes or gene products can be modulatedand the plants tested by screening for the desired trait. Specifically,the gene, mRNA levels, or protein levels can be altered in a plantutilizing the procedures described herein and the phenotypes can beassayed. As an example, a plant can be transformed according to Bechtoldand Pelletier (1998, Methods. Mol. Biol. 82:259-266) and/or screened forvariants as in Winkler et al. (1998) Plant Physiol 118: 743-50 andvisually inspected for the desired phenotype or metabolically and/orfunctionally assayed according to de Castro (1998, Phytochemistry 47:689-694), Xu (1998, J Exp Bot 49: 573-582), Ausubel et al. (In: CurrentProtocols in Molecular Biology (1999) Volume 1, chapter 4, eds. Ausubel,Brent, Kingston, Moore, Seidman, Smith and Struhl, New York, N.Y.) andDe Castro et al. (2000, Plant Physiol 122: 327-36)

III.E.9.b. Use of Osmotic Stress Responsive Genes to ModulateBiochemical Activities

The activities of one or more of the osmotic stress responsive genes canbe modulated to change biochemical or metabolic activities and/orpathways such as those noted below. Such biological activities can bemeasured according to the citations included in the Table below:

BIOCHEMICAL OR CITATIONS METABOLIC ACTIVITIES INCLUDING PROCESS AND/ORPATHWAYS ASSAYS Cell Growth And Regulation Of Osmolyte Yoshu et al.(1995) Differentiation Synthesis The Plant Journal 7: 751-60 RegulationOf Glycolate Pathway Streb et al. (1993) And Photoinhibition OfPhysiologia Photosystem II In Response To Plantarum. 88: 590-598 StressGene Regulation Transcriptional Regulation Of Current Protocols inOsmotic Stress Induced Proteins Molecular Biology/edited Through DNABinding Proteins by Frederick M. Ausubel . . . [et al.]. New York:Published by Greene Pub. Associates and Wiley- Interscience: J. Wiley,c1987 Transcriptional Regulation Of Jonak (1996) Proceedings OsmoticStress Induced Proteins of the National Academy of Through ProteinPhosphorylation Sciences of the United And Dephosphorylation States ofAmerica, 93: 11274-11279; Monroy, A. et al., (1998) AnalyticalBiochemistry 265: 183-185; Regulation Of Osmotic Stress McCright (1998)IN: Induced Gene Protein Methods in Molecular Accumulation By ProteinProtein Biology; Protein Intereaction Between Osmotic phosphataseprotocols; Stress Regulated Genes And Ludlow (1998) Humana ProteinPhosphatase 2C Press Inc.; Suite 808, 999 Riverview Drive, Totowa, NewJersey 07512, USA.: 263-277. Transcriptional Regulation Of Luo and Dean(1999) Heat Induced Genes Through Journal of the Chromatin RemodelingNational Cancer Institute 91: 1288-1294; Chromatin protocols (1999)edited by Peter B. Becker. Totowa, N.J.: Humana Press Activity OfAbcisic Acid Gubler et al. (1999) Regulated DNA Binding Proteins PlantJournal 17: 1-9 Accumulation Of RNA Binding Sato (1995) Proteins ThatRegulate Osmotic Nucleic Acids Research 23: Stress 2161-2167. StressResponse Synthesis And Metabolism Of Minocha et al. Osmoprotectants SuchAs (1999) Plant Betaine, Proline And Trehalase Physiol and Biochem 37:597-603 Regulation Of Sugar Transporters Dejardin et al. (1999) BiochemJ; 344 Pt 2: 503-9 Regulation Of Vacuolar Gaxiola et al. Sodium/ProtonAntiport Activity (1999) PNAS USA And The Detoxification Of 96:1480-1485 Cations Regulation Of Intracellular Na+ Espinoza-Ruiz et AndLi+ Ion Concentrations al. (1999) The Plant Journal 20: 529-539Regulation Of Universal Stress Freestone et al. Protein HomologueActivity By (1997) Journal of Phosphorylation And Molecular Biology,Dephosphorylation. v. 274: 318-324 Regulation/Maintenance Of Walker(1996) Protein Stability During Thermal Humana Press Inc. Stress Suite808, 999 Riverview Drive, Totowa, New Jersey 07512, USA Regulation OfProtein Vierstra (1996) Plant Degradation During Thermal MolecularBiology, 32: 275-302. Stress. Vierstra and Callis (1999) Plant MolecularBiology, 41: 435-442 Signal Transduction Activation Of Stress ResponseXinong et al. Genes (1999) The Plant Journal 19: 569-578 Salt TolerancePiao (1999) Plant Physiol 19: 1527-1534 Calcium Mediated Stress Subbaiahet al. Response (1994) Plant Physiology 105: 369-376 Kudla et al. (1999)PNAS USA 96: 4718-4723

Other biological activities that can be modulated by the osmotic stressresponsive genes and gene products are listed in the Reference tables.Assays for detecting such biological activities are described in theProtein Domain table.

Osmotic stress responsive genes are characteristically differentiallytranscribed in response to fluctuating osmotic stress levels orconcentrations, whether internal or external to an organism or cell.MA_diff table reports the changes in transcript levels of variousosmotic stress responsive genes in aerial tissues of plants at 1 and 6hours after the plants were sprayed with Hoagland's solution containing20% PEG as compared to aerial tissues from plants sprayed withHoagland's solution only.

The data from this time course can be used to identify a number of typesof osmotic stress responsive genes and gene products, including “earlyresponding,” “sustained osmotic stress responders,” “repressors ofosmotic stress pathways” and “osmotic stress responders.” Profiles ofthese different osmotic stress responsive genes are shown in the Tablebelow together with examples of the kinds of associated biologicalactivities.

EXAMPLES OF GENE FUNCTIONAL BIOCHEMICAL EXPRESSION CATEGORY OFPHYSIOLOGICAL ACTIVITIES OF LEVELS GENES CONSEQUENCES GENE PRODUCTS UpRegulated Early Responders Osmotic Stress Transcription Transcripts ToOsmotic Perception Factors (Level At 1 Hour ≅ Stress Osmolyte UptakeTranscription 6 Hours) Universal Stress Modulation Of Coactivators(Level At 1 Hour > Response Genes Osmotic Stress Membrane 6 Hours)Osmotic Stress Response Signal Transporters Responders TransductionPathways Proline Abscisic Acid Specific Gene Biosynthesis BiosynthesisAnd Transcription Selective Inhibition Perception Initiation Of OsmolyteSpecific Gene Transport Transcription Protein Repression UbiquitinationTranslation Activation Protein Translation Degradation Repression RnaBinding Repression Of Proteins “Normal State” Modification Of PathwaysTo Optimize Protein Activity By Osmotic Stress Phosphatases, ResponseKinases Activation Of Stress Synthesis And Or Signaling PathwaysActivation Of Up Regulation Of Oxide Hydrolases, Abscisic AcidSuoeroxidedismutase, Biosynthesis Pathway Iron Ascorbate ProteinAccumulation Peroxidase And Activity Activation Of Scavenging ReactiveSignaling Pathway Oxygen Species By Calcium Modification Of Cell BindingProteins, Wall Composition Modification Of Up-Regulation Of ProteinActivity By Universal Stress Protein-Protein Response ProteinInteraction Accumulation Change In Chromatin Structure And/Or LocalizedDna Topology Modification Of Pre-Existing Translation Factors ByPhosphorylation (Kinases) Or Dephosphorylation (Phosphatases) SynthesisOf New Translation Factors Abscisic Acid Biosynthesis Up RegulatedSustained Osmolyte Adjustment Osmotic Stress Transcripts Osmotic StressAnd Adaptation Metabolic (Level At 1 Hr < 6 Hr) RespondersPhotosynthetic Pathways Repressor Of Activity Modification SugarBiosynthetic Osmotic Stress Activation Of Pathways Pathways “NormalState” Sugar Transporters Abscisic Acid Biosynthesis Genes TranscriptionPerception, Negative Regulation Factors Biosynthesis And Of OsmoticStress Transcription Regulation Pathways Coactivators NegativeRegulation Membrane Of Abscisic Acid Transporters Biosynthesis AbscisicAcid Acivation Of Abscisic Biosynthesis Acid Degradation Pathway CellWall Composition Modification Down-Regulated Early Responder MetabolicRepression Transcription Transcripts Repressors Of Specific Gene Factors(Level At 1 Hr ≈ 6 Hr) “Normal” State Of Transcription Transcription(Level At 6 Hr > 1 Hr) Metabolism Initiation Coactivators NegativeRegulators Specific Gene Protein Of Abscisic Acid TranscriptionDegradation Biosynthesis And Repression Rna Binding Perception.Translation Activation Proteins Positive Regulators TranslationModification Of Of “Normal State” Repression Protein Activity ByMetabolic Pathways. Abscisic Acid Phosphatases, Degradation KinasesProtein Degradation Activation Of Signaling Pathway By Calcium BindingProteins, Modification Of Protein Activity By Protein-ProteinInteraction Change In Chromatin Structure And/Or Localized Dna TopologyModification Of Pre-Existing Translation Factors By Phosphorylation(Kinases) Or Dephosphorylation (Phosphatases) Synthesis Of NewTranslation Factors Down-Regulated Repressors Of Osmotic StressTranscription Transcripts “Normal” State Of Adaptation Factors (Level At1 Hr > 6 Hr) Metabolism Negative Regulation Transcription Genes With OfAbscisic Acid Coactivators Discontinued Biosynthesis Protein ExpressionOr Negative Regulation Degradation UnsTable mRNA In Of Osmotic StressRna Binding Presence Of Osmotic Response Pathways Proteins Stress GenesModification Of Repressor Of Osmolyte Synthesis Protein Activity ByOsmotic Stress And Osmolyte Phosphatases, Pathways Cellular PartitioningKinases Repressors Of Readjustment Activation Of Abscisic AcidActivation Of Signaling Pathway Biosynthesis, “Normal State” By CalciumPerception And Metabolic Pathways Binding Proteins, RegulationModification Of Protein Activity By Protein-Protein Interaction ChangeIn Chromatin Structure And/Or Localized Dna Topology Modification OfPre-Existing Translation Factors By Phosphorylation (Kinases) OrDephosphorylation (Phosphatases) Synthesis Of New Translation FactorsSugar Biosynthetic Pathways Sugar Transporters

Further, any desired sequence can be transcribed in similar temporal,tissue, or environmentally specific patterns as the osmotic stressresponsive genes when the desired sequence is operably linked to apromoter of an osmotic stress responsive gene.

III.E.10. Aluminum Responsive Genes, Gene Components and Products

Aluminum is toxic to plants in soluble form (Al³⁺). Plants grown underaluminum stress have inhibited root growth and function due to reducedcell elongation, inhibited cell division and metabolic interference. Asan example, protein inactivation frequently results from displacement ofthe Mg2+ cofactor with aluminum. These types of consequences result inpoor nutrient and water uptake. In addition, because stress perceptionand response occur in the root apex, aluminum exposure leads to therelease of organic acids, such as citrate, from the root as the plantattempts to prevent aluminum uptake.

The ability to endure soluble aluminum is a major determinant of thegeographical distribution and productivity of agricultural crops.Decreases in yield and crop failure frequently occur as a result ofaberrant, hot conditions even in areas considered suitable for thecultivation of a given species or cultivar. Only modest increases in thealuminum tolerance of crop species would have a dramatic impact onagricultural productivity. The development of genotypes with increasedaluminum tolerance would provide a more reliable means to minimize croplosses and diminish the use of costly practices to modify theenvironment.

Microarray technology allows monitoring of gene expression levels forthousands of genes in a single experiment. This is achieved bysimultaneously hybridizing two differentially labeled fluorescent cDNApools to glass slides that contain spots of DNA (Schena et al. (1995)Science 270: 467-70). The Arabidopsis Functional Genomics Consortium(AFGC) has recently made public the results from such microarrayexperiments conducted with AFGC chips containing 10,000 non-redundantESTs, selected from 37,000 randomly sequenced ESTs generated from mRNAof different tissues and developmental stages.

The sequences of the ESTs showing at least two-fold increases ordecreases over the controls were identified, compared to the Ceresfull-length cDNA and genomic sequence databanks, and identical Ceresclones identified. MA_diff table reports the results of this analysis,indicating those Ceres clones which are up or down regulated overcontrols, thereby indicating the Ceres clones which are aluminumresponse responsive genes.

The MA_diff Table(s) reports the transcript levels of the experiment(see EXPT ID: Aluminum (relating to SMD 7304, SMD 7305)). Fortranscripts that had higher levels in the samples than the control, a“+” is shown. A “−” is shown for when transcript levels were reduced inroot tips as compared to the control. For more experimental detail seethe Example section below.

Aluminum genes are those sequences that showed differential expressionas compared to controls, namely those sequences identified in theMA_diff tables with a “+” or “−” indication.

Aluminum Genes Identified by Cluster Analyses of Differential Expression

Aluminum Genes Identified by Correlation to Genes that areDifferentially Expressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of Aluminum genes is any group in the MA_clust thatcomprises a cDNA ID that also appears in Expt ID Aluminum (relating toSMD 7304, SMD 7305) of the MA_diff table(s).

Aluminum Genes Identified by Correlation to Genes that CausePhysiological Consequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of Aluminumgenes. A group in the MA_clust is considered a Aluminum pathway ornetwork if the group comprises a cDNA ID that also appears in Knock-inor Knock-out tables that causes one or more of the phenotypes describedin section above.

Aluminum Genes Identified by Amino Acid Sequence Similarity

Aluminum genes from other plant species typically encode polypeptidesthat share amino acid similarity to the sequences encoded by corn andArabidopsis Aluminum genes. Groups of Aluminum genes are identified inthe Protein Group table. In this table, any protein group that comprisesa peptide ID that corresponds to a cDNA ID member of a Aluminum pathwayor network is a group of proteins that also exhibits Aluminumfunctions/utilities.

III.E.10.a. Use of Aluminum Response Genes to Modulate Phenotypes

Changes in aluminum concentrations in a plant's surrounding environmentresults in modulation of many genes and gene products. Examples of suchaluminum response genes and gene products are shown in the Reference andSequence Tables. These genes and/or products are responsible for effectson traits such as plant vigor and seed yield.

While aluminum responsive polynucleotides and gene products can actalone, combinations of these polynucleotides also affect growth anddevelopment. Useful combinations include different aluminum responsivepolynucleotides and/or gene products that have similar transcriptionprofiles or similar biological activities, and members of the same orsimilar biochemical pathways. In addition, the combination of a aluminumresponsive polynucleotide and/or gene product with anotherenvironmentally responsive polynucleotide is also useful because of theinteractions that exist between hormone-regulated pathways, stresspathways, nutritional pathways and development. Here, in addition topolynucleotides having similar transcription profiles and/or biologicalactivities, useful combinations include polynucleotides that may havedifferent transcription profiles but which participate in a commonpathway.

Such aluminum responsive genes and gene products can function to eitherincrease or dampen the above phenotypes or activities either

-   -   in response to changes in aluminum concentration or    -   in the absence of aluminum fluctuations.

More specifically, aluminum responsive genes and gene products areuseful to or modulate one or more phenotypes including growth; roots(such as inhibition of root elongation); stems; leaves; whole plant;development (such as cell growth, elongation, and division) and mediatesresponse to oxidative stress, calcium-mediated defense, antioxidantdefense and pathogenesis.

To produce the desired phenotype(s) above, one or more of the aluminumresponse genes or gene products can be tested by screening for thedesired trait. Specifically, the gene, mRNA levels, or protein levelscan be altered in a plant utilizing the procedures described herein andthe phenotypes can be assayed. As an example, a plant can be transformedaccording to Bechtold and Pelletier (1998, Methods. Mol. Biol.82:259-266) and visually inspected for the desired phenotype ormetabolically and/or functionally assayed according to Li and Fleming(1999, FEBS Lett 461: 1-5), Delhaize et al. (1999, J Biol Chem 274:7082-8), Sigimoto and Sakamoto (1997, Genes Genet Syst 72: 311-6), Esakiet al. (2000, Plant Physiol 122: 657-65), Leonard and Gerber (1988,Mutat Res 196: 247-57), Baisakhi et al. (2000, Mutat Res 465: 1-9), Ma(2000, Plant Cell Physiol 41: 383-90) and Koyama et al. (1999, PlantCell 40: 482-8)

Alternatively, the activities of one or more of the aluminum responsivegenes can be modulated to change biochemical or metabolic activitiesand/or pathways such as those noted below. Such biological activitiescan be measured according to the citations included in the Table below:

BIOCHEMICAL OR METABOLIC GENERAL ACTIVITIES AND/OR CATEGORY PATHWAYSASSAY Cell Growth and Phospholipase D (PLD) Toda et al. (1999)Development activity Biosci Biotechnol Biochem 63: 210-212 Regulation ofPhosphtidylserine Synthase (PSS) Cell wall strengthening Hamel et al.(1998) Planta 205: 531-38 Stress Response Regulation of oxidative stressEsaki et al. (2000) Plant Physiol 122: 657-655 Regulation of Baisakhi etal. antioxidant defense and (2000) Mutat Res DNA repair 465: 1-9Secretion of Organic Koyama et al. Acids (e.g. maleate, (1999) PlantCell citrate) from root apex 40: 482-8 Ca2+mediated Defense Plieth etal. (1999) Responses Against Low Plant J 18: 634-50 pH Signaling H+transport Degenhardt et al. (1988) Plant Physil 117: 19-27 Auxintransport Rashotte et al. (2000) Plant Physiol 122: 481-90

Other biological activities that can be modulated by aluminum responsegenes and their products are listed in the REFERENCE Table. Assays fordetecting such biological activities are described in the Protein Domaintable.

EXAMPLES OF TRANSCRIPT PHYSIOLOGICAL BIOCHEMICAL LEVELS TYPE OF GENESCONSEQUENCES ACTIVITY Up regulated responders to Aluminum Transporterstranscripts aluminum perception Metabolic enzymes application Aluminumuptake Change in cell and transport membrane structure Aluminum andpotential metabolism Kinases and Synthesis of phosphatases secondaryTranscription metabolites and/or activators proteins Change in chromatinModulation of structure and/or aluminum localized DNA response topologytransduction pathways Specific gene transcription initiationDown-regulated responder to Negative Transcription factors transcriptsaluminum regulation of Change in protein repressors of aluminumstructure by aluminum state of pathways phosphorylation metabolismChanges in (kinases) or Genes with pathways and dephosphorylationdiscontinued processes (phosphatases) expression or operating in cellsChange in chromatin unsTable mRNA in Changes in other structure and/orDNA presence of aluminum metabolisms than topology aluminum Stability offactors for protein synthesis and degradation Metabolic enzymes

Use of Promoters of Aluminum Responsive Genes

Promoters of Aluminum responsive genes are useful for transcription ofany desired polynucleotide or plant or non-plant origin. Further, anydesired sequence can be transcribed in a similar temporal, tissue, orenvironmentally specific patterns as the Aluminum responsive genes wherethe desired sequence is operably linked to a promoter of a Aluminumresponsive gene. The protein product of such a polynucleotide is usuallysynthesized in the same cells, in response to the same stimuli as theprotein product of the gene from which the promoter was derived. Suchpromoter are also useful to produce antisense mRNAs to down-regulate theproduct of proteins, or to produce sense mRNAs to down-regulate mRNAsvia sense suppression.

III.E.11. Cadmium Responsive Genes, Gene Components and Products

Cadmium (Cd) has both toxic and non-toxic effects on plants. Plantsexposed to non-toxic concentrations of cadmium are blocked for viraldisease due to the inhibition of systemic movement of the virus.Surprisingly, higher, toxic levels of Cd do not inhibit viral systemicmovement, suggesting that cellular factors that interfere with the viralmovement are triggered by non-toxic Cd concentrations but repressed inhigh Cd concentrations. Furthermore, exposure to non-toxic Cd levelsappears to reverse posttranslational gene silencing, an inherent plantdefense mechanism. Consequently, exploring the effects of Cd exposurehas potential for advances in plant disease control in addition to soilbio-remediation and the improvement of plant performance in agriculture.

Changes in cadmium concentrations in a plant's surrounding environmentresults in modulation of many genes and gene products. Microarraytechnology allows monitoring of gene expression levels for thousands ofgenes in a single experiment. This is achieved by simultaneouslyhybridizing two differentially labeled fluorescent cDNA pools to glassslides that contain spots of DNA (Schena et al. (1995) Science 270:467-70). The US Arabidopsis Functional Genomics Consortium (AFGC) hasrecently made public the results from such microarray experimentsconducted with AFGC chips containing some 10,000 non-redundant ESTs,selected from about 37,000 randomly sequenced ESTs generated from mRNAof different tissues and developmental stages.

The sequences of the ESTs showing at least two-fold increases ordecreases in plants treated with 10 μM cadmium compared with untreatedplants were identified, compared to the Ceres full length cDNA andgenomic sequence databanks, and the equivalent Ceres clones identified.The MA_diff table(s) report(s) the results of this analysis, indicatingthose Ceres clones which are up or down regulated over controls, therebyindicating the Ceres clones which represent cadmium responsive genes.

Examples of such cadmium responsive genes and gene products are shown inthe Reference and Sequence Tables. These genes and/or products areresponsible for effects on traits such as plant vigor and seed yield.

While cadmium responsive polynucleotides and gene products can actalone, combinations of these polynucleotides also affect growth anddevelopment. Useful combinations include different cadmium responsivepolynucleotides and/or gene products that have similar transcriptionprofiles or similar biological activities, and members of the same orsimilar biochemical pathways. Whole pathways or segments of pathways arecontrolled by transcription factor proteins and proteins controlling theactivity of signal transduction pathways. Therefore, manipulation ofsuch protein levels is especially useful for altering phenotypes andbiochemical activities of plants. In addition, the combination of acadmium responsive polynucleotide and/or gene product with otherenvironmentally responsive polynucleotides is also useful because of theinteractions that exist between, for example, stress and pathogeninduced pathways, nutritional pathways and development. Here, inaddition to polynucleotides having similar transcription profiles and/orbiological activities, useful combinations include polynucleotides thatmay have different transcription profiles but which participate incommon or overlapping pathways.

The MA_diff Table(s) reports the transcript levels of the experiment(see EXPT ID: Cadium (relating to SMD 7427, SMD 7428)). For transcriptsthat had higher levels in the samples than the control, a “+” is shown.A “−” is shown for when transcript levels were reduced in root tips ascompared to the control. For more experimental detail see the Examplesection below.

Cadium genes are those sequences that showed differential expression ascompared to controls, namely those sequences identified in the MA_difftables with a “+” or “−” indication.

Cadium Genes Identified by Cluster Analyses of Differential Expression

Cadium Genes Identified by Correlation to Genes that are DifferentiallyExpressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of Cadium genes is any group in the MA_clust thatcomprises a cDNA ID that also appears in Expt ID Cadium (relating to SMD7427, SMD 7428) of the MA_diff table(s).

Cadium Genes Identified by Correlation to Genes that Cause PhysiologicalConsequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of Cadiumgenes. A group in the MA_clust is considered a Cadium pathway or networkif the group comprises a cDNA ID that also appears in Knock-in orKnock-out tables that causes one or more of the phenotypes described insection above.

Cadium Genes Identified by Amino Acid Sequence Similarity

Cadium genes from other plant species typically encode polypeptides thatshare amino acid similarity to the sequences encoded by corn andArabidopsis Cadium genes. Groups of Cadium genes are identified in theProtein Group table. In this table, any protein group that comprises apeptide ID that corresponds to a cDNA ID member of a Cadium pathway ornetwork is a group of proteins that also exhibits Cadiumfunctions/utilities.

Such cadmium responsive genes and gene products can function to eitherincrease or dampen phenotypes or activities either in response tochanges in cadmium concentration or in the absence of cadmiumfluctuations. Further, promoters of cadmium responsive genes, asdescribed in the Reference tables, for example, are useful to modulatetranscription that is induced by cadmium or any of the followingphenotypes or biological activities below.

III.E.11.a. Use of Cadmium Responsive Genes, Gene Components andProducts to Modulate Phenotypes

Cadmium responsive genes and gene products are useful to or modulate oneor more phenotypes including growth, roots, initiation and maintenanceof cell division, stems, leaves, development, mitochondria,post-embryonic root meristem development, senescence, stress response,modulation of jasmonic acid and other stress control pathways, metabolicdetoxification, heavy metals, plant and seed yield; and fruit yield.

Further, any desired sequence can be transcribed in similar temporal,tissue, or environmentally specific patterns as the cadmium responsivegenes when the desired sequence is operably linked to a promoter of acadmium responsive gene.

To regulate any of the phenotype(s) above, activities of one or more ofthe cadmium responsive genes or gene products can be modulated andtested by screening for the desired trait. Specifically, the gene, mRNAlevels, or protein levels can be altered in a plant utilizing theprocedures described herein and the phenotypes can be assayed. As anexample, a plant can be transformed according to Bechtold and Pelletier(1998) Methods. Mol. Biol. 82:259-266) and/or screened for variants asin Winkler et al. (1998) Plant Physiol 118: 743-50 and visuallyinspected for the desired phenotype or metabolically and/or functionallyassayed according to Ghoshroy et al. (1998, Plant J 13: 591-602),Citovsky et al. (1998, Plant J 16: 13-20), Clemens et al. (1999, EMBO J18: 3325-33), Chen et al. (2000, Chemosphere 41: 229-34), Xian andOliver (1998, Plant Cell 10: 1539-90), Romero-Peurtas et al. (1999, FreeRad Res 31: S25-31), Gaur and Noraho (1995, Biomed Environ Sci 8:202-10), Thomine et al. (2000, PNAS USA 97: 4991-6), Howden et al.(1995, Plant Physiol 107: 1067-73), Kesseler and Brand (1994, Eur JBiochem 225: 907-22) and Vernoux et al. (2000, Plant Cell 12: 97-110).

III.E.10.b. Use of Cadmium-Responsive Genes, Gene Components andProducts to Modulate Biochemical Activities

The activities of one or more of the cadmium responsive genes can bemodulated to change biochemical or metabolic activities and/or pathwayssuch as those noted below. Such biological activities can be measuredaccording to the citations included in the Table below:

BIOCHEMICAL OR METABOLIC ACTIVITIES CITATIONS INCLUDING PROCESS AND/ORPATHWAYS ASSAYS Growth, Differentiation Root Growth Thomine et al.(2000) PNAS and Development Initiation and maintenance of USA 97: 4991-6cell division Vernoux et al. (2000) Plant Cell Resistance to Cadmium-12: 97-110 inhibition of root growth Metabolism Cadmium sensing Howdenet al. (1995) Plant Physiol 107: 1067-73 Cadmium uptake and Gaur andNoraho (1995) Biomed transport Environ Sci 8: 202-10 Decreased cadmiumThomine et al. (2000) PNAS transport USA 97: 4991-6 PhytoremediationInhibition of oxidative Kesseler and Brand (1994) Eur. phophorylationBiochem 225: 907-22 Plant Defenses Viral resistance Ghoshroy et al.(1998) Plant J Inhibition of systemic 13: 591-602 movement of virusBlock of viral disease Detoxification of heavy Clemens et al. (1999)EMBO J metals 18: 3325-33 Enhanced stress resistance Romero-Peurtas etal. (1999) Free Rad Res 31: S25-31 Cadmium resistance Xiang and Oliver(1998) Plant via modulation of jasmonic Cell 10: 1539-90 acid signalingpathway Signaling Relief of post-translational Citovsky et al. (1998)Plant J 16: gene silencing 13-20

Other biological activities that can be modulated by the cadmiumresponsive genes and gene products are listed in the Reference tables.Assays for detecting such biological activities are described in theProtein Domain table.

Cadmium responsive genes are characteristically differentiallytranscribed in response to fluctuating cadmium levels or concentrations,whether internal or external to an organism or cell. The MA_difftable(s) report(s) the changes in transcript levels of various cadmiumresponsive genes following treatment with 10 μM cadmium, relative tountreated plants.

Profiles of some cadmium responsive genes are shown in the Table belowtogether with examples of the kinds of associated biological activities.

EXAMPLES OF TRANSCRIPT TYPE OF PHYSIOLOGICAL BIOCHEMICAL LEVELS GENESCONSEQUENCES ACTIVITY Up regulated Responders to Cadmium perceptionTransporters transcripts cadmium Cadmium uptake and Metabolic enzymesApplication transport Change in cell membrane Genes induced by Cadmiummetabolism structure and potential cadmium Synthesis of secondaryKinases and metabolites and/or Phosphatases proteins Transcriptionactivators Modulation of Change in chromatin cadmium response structureand/or localized transduction pathways DNA topology Specific gene RNAbinding proteins transcription initiation Genes involved in inhibitingsystemic movement of plant viral RNA Genes involved in posttranslational gene silencing Down-regulated Responders to Negativeregulation of Transcription factors transcripts cadmium cadmium pathwaysChange in protein Genes repressed released structure by by cadmiumChanges in pathways phosphorylation Genes with and processes operating(kinases) or discontinued in cells Dephosphoryaltion expression orChanges in metabolism (phosphatases) unsTable mRNA other than cadmiumChange in chromatin in presence of pathways structure and/or DNA cadmiumGenes involved in topology facilitating systemic Factors for proteinmovement of plant synthesis and degradation viral RNA Metabolic enzymesGenes involved in RNA binding proteins promoting post translational genesilencing

Use of Promoters of Cadmium Responsive Genes

Promoters of Cadmium responsive genes are useful for transcription ofany desired polynucleotide or plant or non-plant origin. Further, anydesired sequence can be transcribed in a similar temporal, tissue, orenvironmentally specific patterns as the Cadmium responsive genes wherethe desired sequence is operably linked to a promoter of a Cadmiumresponsive gene. The protein product of such a polynucleotide is usuallysynthesized in the same cells, in response to the same stimuli as theprotein product of the gene from which the promoter was derived. Suchpromoter are also useful to produce antisense mRNAs to down-regulate theproduct of proteins, or to produce sense mRNAs to down-regulate mRNAsvia sense suppression.

III.12. Disease Responsive Genes, Gene Components and Products

Often growth and yield are limited by the ability of a plant to toleratestress conditions, including pathogen attack. To combat such conditions,plant cells deploy a battery of inducible defense responses, includingthe triggering of an oxidative burst and the transcription ofpathogenesis-related protein (PR protein) genes. These responses dependon the recognition of a microbial avirulence gene product (avr) by aplant resistance gene product (R), and a series of downstream signalingevents leading to transcription-independent and transcription-dependentdisease resistance responses. Reactive oxygen species (ROS) such as H₂O₂and NO from the oxidative burst plays a signaling role, includinginitiation of the hypersensitive response (HR) and induction of systemicacquired resistance (SAR) to secondary infection by unrelated pathogens.PR proteins are able to degrade the cell walls of invadingmicroorganisms, and phytoalexins are directly microbicidal.

The presence of an avirulent pathogen and/or changes in theconcentrations of O₂ ⁻, H₂O₂ and NO in the environment surrounding aplant cell modulate the activities of many genes and, therefore, thelevels of many gene products. Examples of tobacco mosaic virus (TMV)responsive genes and gene products, many of them operating through anROS signaling system, are shown in The Reference and Sequence Tables.These genes and/or products are responsible for effects on traits suchas plant vigor and seed yield. The genes were discovered andcharacterized from a much larger set by experiments designed to findgenes whose mRNA products changed in response to application of TMV toplants.

Microarray technology allows monitoring of gene expression levels forthousands of genes in a single experiment. This is achieved byhybridizing labeled fluorescent cDNA pools to glass slides that containspots of DNA (Schena et al. (1995) Science 270: 467-70). The USArabidopsis Functional Genomics Consortium (AFGC) has recently madepublic the results from such microarray experiments conducted with AFGCchips containing some 10,000 non-redundant ESTs, selected from about37,000 randomly sequenced ESTs generated from mRNA of different tissuesand developmental stages.

The sequences of the ESTs showing at least two-fold increases ordecreases in response to TMV infection over the non infected controlswere identified, compared to the Ceres full length cDNA and genomicsequence databanks, and equivalent Ceres clones identified. The MA_difftable(s) report(s) the results of this analysis, indicating those Ceresclones which are up or down regulated over controls, thereby indicatingthe Ceres clones which represent disease responsive genes.

Manipulation of one or more disease responsive gene activities is usefulto modulate the biological processes and/or phenotypes listed below.Disease responsive genes and gene products can act alone or incombination. Useful combinations include disease responsive genes and/orgene products with similar transcription profiles, similar biologicalactivities, or members of the same or functionally related biochemicalpathways. Whole pathways or segments of pathways are controlled bytranscription factor proteins and proteins controlling the activity ofsignal transduction pathways. Therefore, manipulation of such proteinlevels is especially useful for altering phenotypes and biochemicalactivities of plants.

Such disease responsive genes and gene products can function to eitherincrease or dampen the above phenotypes or activities either in responseto changes in active oxygen concentration or in the absence of activeoxygen fluctuations. The MA_diff Table(s) reports the transcript levelsof the experiment (see EXPT ID: Disease (relating to SMD 7342, SMD7343)). For transcripts that had higher levels in the samples than thecontrol, a “+” is shown. A “−” is shown for when transcript levels werereduced in root tips as compared to the control. For more experimentaldetail see the Example section below.

Disease genes are those sequences that showed differential expression ascompared to controls, namely those sequences identified in the MA_difftables with a “+” or “−” indication.

Disease Genes Identified by Cluster Analyses of Differential Expression

Disease Genes Identified by Correlation to Genes that are DifferentiallyExpressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of Disease genes is any group in the MA_clust thatcomprises a cDNA ID that also appears in Expt ID Disease (relating toSMD 7342, SMD 7343) of the MA_diff table(s).

Disease Genes Identified by Correlation to Genes that CausePhysiological Consequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of Diseasegenes. A group in the MA_clust is considered a Disease pathway ornetwork if the group comprises a cDNA ID that also appears in Knock-inor Knock-out tables that causes one or more of the phenotypes describedin section above.

Disease Genes Identified by Amino Acid Sequence Similarity

Disease genes from other plant species typically encode polypeptidesthat share amino acid similarity to the sequences encoded by corn andArabidopsis Disease genes. Groups of Disease genes are identified in theProtein Group table. In this table, any protein group that comprises apeptide ID that corresponds to a cDNA ID member of a Disease pathway ornetwork is a group of proteins that also exhibits Diseasefunctions/utilities.

Further, promoters of disease responsive genes, as described in theReference tables, for example, are useful to modulate transcription thatis induced by disease or any of the following phenotypes or biologicalactivities below. Further, any desired sequence can be transcribed insimilar temporal, tissue, or environmentally specific patterns as thedisease responsive genes when the desired sequence is operably linked toa promoter of a disease responsive gene.

III.E.12.a. Use of Disease Responsive Genes, Gene Components andProducts to Modulate Phenotypes

Disease responsive genes and gene products are useful to or modulate oneor more phenotypes including pathogen tolerance and/or resistance; Avr/Rlocus interactions; non-host interactions; HR; SAR; resistance tobacteria e.g. to Erwinia stewartii, Pseudomonas syringae, Pseudomonastabaci, Stuart's wilt, etc.; resistance to fungi, e.g. to downy mildewssuch as Scleropthora macrospora, Sclerophthora rayissiae, Sclerosporagraminicola, Peronosclerospora sorghi, Peronosclerospora philippinensis,Peronosclerospora sacchari, Peronosclerospora maydis; rusts such asPuccinia sorphi, Puccinia polysora, Physopella zeae, etc.; and to otherfungal diseases e.g. Cercospora zeae-maydis, Colletotrichum graminicola,Fusarium monoliforme, Exserohilum turcicum, Bipolaris maydis,Phytophthora parasitica, Peronospora tabacina, Septoria, etc.;resistance to viruses or viroids e.g. to tobacco or cucumber mosaicvirus, ringspot virus, necrosis virus, pelargonium leaf curl virus, redclover mottle virus, tomato bushy stunt virus, and like viruses;rrResistance to insects, such as to aphids e.g. Myzus persicae; tobeetles and beetle larvae; to lepidoptera larvae, e.g. Heliothus etc.;resistance to Nematodes, e.g. Meloidogyne incognita etc.; localresistance in primary (infected) or secondary (uninfected) leaves;stress tolerance; winter survival; cold tolerance; salt tolerance; heavymetal tolerance, such as cadmium; tolerance to physical wounding;increased organelle tolerance to redox stress, such as in mitochondria,and chloroplasts; cell death; programmed cell death, including death ofdiseased tissue and during senescence; fruit drop; biomass; fresh anddry weight during any time in plant life, such as maturation; number offlowers, seeds, branches, and/or leaves; seed yield, including number,size, weight, and/or harvest index; fruit yield, including number, size,weight, and/or harvest index; plant development; time to fruit maturity;cell wall strengthening and reinforcement; plant product quality; papermaking quality; food additives; treatment of indications modulated byfree radicals; cancer; kinds of low molecular weight compounds such asphytoalexins; abundance of low molecular weight compounds such asphytoalexins; other phenotypes based on gene silencing.

To regulate any of the phenotype(s) above, activities of one or more ofthe disease responsive genes or gene products can be modulated and theplants can be tested by screening for the desired trait. Specifically,the gene, mRNA levels, or protein levels can be altered in a plantutilizing the procedures described herein and the phenotypes can bescreened for variants as in Winkler et al. (1998) Plant Physiol 118:743-50 and assayed, for example, in accordance to Alvarez et al., (1998)Cell 92: 773-784; Halhbrock and Scheel, (1989) Ann. Rev. Plant Physiol.Plant Mol. Biol. 40: 347-369; Lamb et al., (1997) Ann. Rev. Plant Mol.Biol. Plant Physiol. 48: 251-275; Lapwood et al. (1984) Plant Pathol.33: 13-20; Levine et al. (1996) Curr. Biol. 6: 427-437; McKersie et al.,(2000) Plant Physiol. 122: 1427-1437; Olson and Varner (1993) Plant J.4: 887-892; Pastore et al., (2000), FEBS Lett 470: 88-92; Pastori etal., (1997) Plant Physiol. 113: 411-418; Romero-Puertas et al., (1999)Free Radic. Res. 1999 31 Suppl: S25-31; Shirataki et al., Anticancer Res20: 423-426 (2000); Wu et al., (1995) Plant Cell 7: 1357-1368.

III.E.12.b. Use of Disease Responsive Genes, Gene Components andProducts to Modulate Biochemical Activities

The activities of one or more of the disease responsive genes can bemodulated to change biochemical or metabolic activities and/or pathwayssuch as those noted below. Such biological activities are documented andcan be measured according to the citations above and included in theTable below:

BIOCHEMICAL OR METABOLIC ACTIVITIES CITATIONS INCLUDING PROCESS AND/ORPATHWAYS ASSAYS Resistance to Pathogens Induction of ROS signaling Wuet. al. (1995) Plant Cell 7: pathways 1357-68 Modulation of nitric oxideDelledonne et al. (1998) Nature signaling 394: 585-588 Induction of PRproteins, Chamnongpol et. al. (1998) Proc. phytoalexins, and defenseNat. Acad Sci USA 12; 95: 5818-23. pathways Davis et al. (1993)Phytochemistry 32: 607-611 Induction of cellular Chen et. al. Plant J.(1996) protectant genes such as 10: 955-966 glutathione S-transferaseGadea et. al. (1999) Mol Gen (GST) and ascorbate Genet 262: 212-219peroxidase Wu et. al. (1995) Plant Cell 7: 1357-68 ROS levels followingOrozco-Cardenas and Ryan wounding and changes in (1999) Proc. Nat. Acad.Sci. USA physical pressure 25; 96: 6553-7. Yahraus et al. (1995) PlantPhysiol. 109: 1259-1266 Salicyclic acid levels and Durner and Klessig(1996) signaling J.Biol. Chem. 271: 28492-501 Responses to WoundingExpression of genes Involved Legendre et al. (1993) Plant in woundrepair and cell Physiol. 102: 233-240 division Responses toEnvironmental Expression of genes involved Shi et al. (2000) Proc. Natl.Acad. Stress in responses to drought, cold, Sci. USA 97: 6896-6901 salt,heavy metals Reinforcement of Cell Walls Modulation of the ProductionBradley et al. (1992) Cell 70, 21-30 of ExtracTable Proline-Rich ProteinModulation of Lignification Mansouri et al. (1999) Physiol. Plant 106:355-362 Programmed Cell Death Induction of PCD activating Levine et al.(1996) Curr. Biol. 6: genes 427-437. Reynolds et. al. (1998) Biochem. J.330: 115-20 Suppression of PCD Pennell and Lamb (1997) Plant suppressinggenes Cell 9, 1157-1168

Other biological activities that can be modulated by the diseaseresponsive genes and their products are listed in the Reference Table.Assays for detecting such biological activities are described in theProtein Domain table.

Disease responsive genes are characteristically differentiallytranscribed in response to fluctuating levels of disease. The MA_difftable(s) report(s) the changes in transcript levels of various diseaseresponsive genes in the aerial parts of a plant 3 days after the plantwas sprayed with a suspension of TMV relative to control plants sprayedwith water.

The data from this experiment reveal a number of types of diseaseresponsive genes and gene products, including “early responders,” and“delayed responders”. Profiles of individual disease responsive genesare shown in the Table below with examples of which associatedbiological activities are modulated when the activities of one or moresuch genes vary in plants.

EXAMPLES OF GENE FUNCTIONAL BIOCHEMICAL EXPRESSION CATEGORYPHYSIOLOGICAL ACTIVITY LEVELS OF GENE CONSequence OF GENE PRODUCTSUpregulated Early ROS Perception and Transcription factors, transcriptsResponders to Response kinases, phosphatases, GTP- Pathogens bindingproteins (G- proteins), leucine rich repeat proteins (LRRs),transporters, calcium binding proteins, chromatin remodeling proteinsInitiation of Gene Glutathione S-transferase Transcription (GST), heatshock proteins, salicylic acid (SA) response pathway proteins, jasmonateresponse pathway proteins, dehydrins, peroxidases, catalases DelayedInitiation of Defence Proteases, pathogen Responders to GeneTranscription response (PR) proteins, Pathogens cellulases, chitinases,cutinases, glucanases, other degrading enzymes, calcium channelblockers, phenylalanine ammonia lyase, proteins of defense pathways,cell wall proteins incuding proline rich proteins and glycine richproteins, epoxide hydrolase, methyl transferases Activation of celldeath Transcription factors pathways kinases, phosphatases, DNAsurveillance proteins, p53, proteases, endonucleases, GTP-bindingproteins (G- proteins), leucine rich repeat proteins (LRRs),transporters, calcium binding proteins, mitochondrial and chloroplastenergy related proteins, ribosome inactivating proteins Initiation ofCellular Reactive oxygen scavenging Protectant Gene enzymes, GST,catalase, Transcription peroxidase, ascorbate oxidase DownregulatedEarly Negative regulation of Transcription factors, transcriptsresponders to pathogen inducible kinases, phosphatases, GTP- pathogenspathways released binding proteins (G- proteins), leucine rich repeatproteins (LRRs), transporters, calcium binding proteins, chromatinremodelling proteins Genes repressed Negative regulation ofTranscription factors, by TMV ROS inducible kinases, phosphatases, GTP-pathways released binding proteins (G- proteins), leucine rich repeatproteins (LRRs), transporters, calcium binding proteins, chromatinremodelling proteins Delayed Negative regulation of Transcriptionfactors, Responders to pathogen inducible kinases, phosphatases, GTP-Pathogens pathways released binding proteins (G- proteins), leucine richrepeat proteins (LRRs), transporters, calcium binding proteins,chromatin remodelling proteins Genes repressed Negative regulation ofTranscription factors, by TMV genes suppressing kinases, phosphatases,GTP- programmed cell death binding proteins (G- released proteins),leucine rich repeat proteins (LRRs), transporters, calcium bindingproteins, chromatin remodelling proteins

Use of Promoters of Disease Responsive Genes

Promoters of Disease responsive genes are useful for transcription ofany desired polynucleotide or plant or non-plant origin. Further, anydesired sequence can be transcribed in a similar temporal, tissue, orenvironmentally specific patterns as the Disease responsive genes wherethe desired sequence is operably linked to a promoter of a Diseaseresponsive gene. The protein product of such a polynucleotide is usuallysynthesized in the same cells, in response to the same stimuli as theprotein product of the gene from which the promoter was derived. Suchpromoter are also useful to produce antisense mRNAs to down-regulate theproduct of proteins, or to produce sense mRNAs to down-regulate mRNAsvia sense suppression.

II.E.13. Defense (LOL2) Responsive Genes, Gene Components and Products

Often growth and yield are limited by the ability of a plant to toleratestress conditions, including pathogen attack. To combat such conditions,plant cells deploy a battery of inducible defense responses, includingthe triggering of an oxidative burst and the transcription ofpathogenesis-related protein (PR protein) genes. Reactive oxygen species(ROS) such as H₂O₂ and NO from the oxidative burst play a signalingrole, including initiation of the hypersensitive response (HR) andinduction of systemic acquired resistance (SAR) to secondary infectionby unrelated pathogens. Some PR proteins are able to degrade the cellwalls of invading microorganisms, and phytoalexins are directlymicrobicidal. Other defense related pathways are regulated by salicylicacid (SA) or methyl jasmonate (MeJ).

These responses depend on the recognition of a microbial avirulence geneproduct (avr) by a plant resistance gene product (R), and a series ofdownstream signaling events leading to transcription-independent andtranscription-dependent disease resistance responses. Current modelssuggest that R− gene-encoded receptors specifically interact withpathogen-encoded ligands to trigger a signal transduction cascade.Several components include ndr1 and eds1 loci. NDR1, EDS1, PR1, as wellas PDF1.2, a MeJ regulated gene and Nim1, a SA regulated gene, aredifferentially regulated in plants with mutations in the LOL2 gene.

LOL2 shares a novel zinc finger motif with LSD1, a negative regulator ofcell death and defense response. Due to an alternative splice site theLOL2 gene encodes two different proteins, one of which contains anadditional, putative DNA binding motif. Northern analysis demonstratedthat LOL2 transcripts containing the additional DNA binding motif arepredominantly upregulated after treatment with both virulent andavirulent Pseudomonas syringae pv maculicola strains. Modulation in thisgene can also confer enhanced resistance to virulent and avirulentPeronospora parasitica isolates

Examples of LOL2 responsive genes and gene products are shown in theReference, Sequence, Protein Group, Protein Group Matrix, MA_diff andMA_clust tables. These genes and/or products are responsible for effectson traits such as plant vigor, disease resistance, and seed yield. Thegenes were discovered and characterized from a much larger set bymicroarray experiments designed to find genes whose mRNA productschanged when the LOL2 gene was mutated in plants.

Microarray technology allows monitoring of gene expression levels forthousands of genes in a single experiment. This is achieved byhybridizing labeled fluorescent cDNA pools to glass slides that containspots of DNA (Schena et al. (1995) Science 270: 467-70). The USArabidopsis Functional Genomics Consortium (AFGC) has recently madepublic the results from such microarray experiments conducted with AFGCchips containing some about 10,000 non-redundant ESTs, selected fromabout 37,000 randomly sequenced ESTs generated from mRNA of differenttissues and developmental stages.

The sequences of the ESTs showing at least two-fold increases ordecreases in plants with the LOL2 mutation versus wildtype wereobtained. Specifically, the plant line lol-2-2 tested, a loss offunction mutation. The ESTs were compared to the Ceres full length cDNAand genomic sequence databanks, and equivalent Ceres clones identified.The MA_diff table reports the results of this analysis, indicating thoseCeres clones which are up or down regulated over controls, therebyindicating the Ceres clones which represent LOL2 responsive genes.

Manipulation of one or more LOL2 responsive gene activities is useful tomodulate the biological processes and/or phenotypes listed below. LOL2responsive genes and gene products can act alone or in combination.Useful combinations include LOL2 responsive genes and/or gene productswith similar transcription profiles, similar biological activities, ormembers of the same or functionally related biochemical pathways. Wholepathways or segments of pathways are controlled by transcription factorproteins and proteins controlling the activity of signal transductionpathways. Therefore, manipulation of such protein levels is especiallyuseful for altering phenotypes and biochemical activities of plants.

Such LOL2 responsive genes and gene products can function to eitherincrease or dampen the above phenotypes or activities either in responseto changes in active LOL2 gene or in the absence. The MA_diff Table(s)reports the transcript levels of the experiment (see EXPT ID: lol2(relating to SMD 8031, SMD 8032)). For transcripts that had higherlevels in the samples than the control, a “+” is shown. A “−” is shownfor when transcript levels were reduced in root tips as compared to thecontrol. For more experimental detail see the Example section below.

Defense genes are those sequences that showed differential expression ascompared to controls, namely those sequences identified in the MA_difftables with a “+” or “−” indication.

Defense Genes Identified by Cluster Analyses of Differential Expression

Defense Genes Identified by Correlation to Genes that are DifferentiallyExpressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of Defense genes is any group in the MA_clust thatcomprises a cDNA ID that also appears in Expt ID lol2 (relating to SMD8031, SMD 8032) of the MA_diff table(s).

Defense Genes Identified by Correlation to Genes that CausePhysiological Consequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of Defensegenes. A group in the MA dust is considered a Defense pathway or networkif the group comprises a cDNA ID that also appears in Knock-in orKnock-out tables that causes one or more of the phenotypes described insection above.

Defense Genes Identified by Amino Acid Sequence Similarity

Defense genes from other plant species typically encode polypeptidesthat share amino acid similarity to the sequences encoded by corn andArabidopsis Defense genes. Groups of Defense genes are identified in theProtein Group table. In this table, any protein group that comprises apeptide ID that corresponds to a cDNA ID member of a Defense pathway ornetwork is a group of proteins that also exhibits Defensefunctions/utilities.

Further, promoters of LOL2 responsive genes, as described in theReference tables, for example, are useful to modulate transcription thatis induced by LOL2 responsive genes or any of the following phenotypesor biological activities below. Further, any desired sequence can betranscribed in similar temporal, tissue, or environmentally specificpatterns as the LOL2 responsive genes when the desired sequence isoperably linked to a promoter of a LOL2 responsive gene.

III.E.12.a. Use of Lol2 Responsive Genes, Gene Components and Productsto Modulate Phenotypes

LOL2 responsive genes and gene products are useful to or modulate one ormore phenotypes including pathogen tolerance and/or resistance; Avr/rlocus interactions; Non-Host interactions; HR; SAR, e.g., diseaseresponsive genes acting in conjunction with infection with any of theorganisms listed below; resistance to bacteria e.g. to Erwiniastewartii, Pseudomonas syringae, Pseudomonas tabaci, Stuart's wilt,etc.; resistance to fungi e.g. to downy mildews such as Scleropthoramacrospora, Sclerophthora rayissiae, Sclerospora graminicola,Peronosclerospora sorghi, Peronosclerospora philippinensis,Peronosclerospora sacchari, Peronosclerospora maydis; rusts such asPuccinia sorphi, Puccinia polysora, Physopella zeae, etc.; and to otherfungal diseases e.g. Cercospora zeae-maydis, Colletotrichum graminicola,Fusarium monoliforme, Exserohilum turcicum, Bipolaris maydis,Phytophthora parasitica, Peronospora tabacina, Septoria, etc.;resistance to viruses or viroids e.g. to tobacco or cucumber mosaicvirus, ringspot virus, necrosis virus, pelargonium leaf curl virus, redclover mottle virus, tomato bushy stunt virus, and like viruses;resistance to insects, such as to aphids e.g. Myzus persicae; to beetlesand beetle larvae; to lepidoptera larvae, e.g. Heliothus etc.;resistance to nematodes, e.g. Meloidogyne incognita etc.; localresistance in primary (infected) or secondary (uninfected) leaves;stress tolerance; winter survival; cold tolerance; salt tolerance, heavymetal tolerance, such as cadmium; tolerance to physical wounding;increased organelle tolerance to redox stress, such as in mitochondria,and chloroplasts; cell death; programmed cell death, including death ofdiseased tissue and during senescence; fruit drop; biomass; fresh anddry weight during any time in plant life, such as maturation; number offlowers, seeds, branches, and/or leaves; seed yield, including number,size, weight, and/or harvest index; fruit yield, including number, size,weight, and/or harvest index; plant development; time to fruit maturity;cell wall strengthening and reinforcement; plant product quality; papermaking quality; food additives; treatment of indications modulated byfree radicals; cancer; kinds of low molecular weight compounds such asphytoalexins; abundance of low molecular weight compounds such asphytoalexins; and other phenotypes based on gene silencing.

To regulate any of the phenotype(s) above, activities of one or more ofthe LOL2 responsive genes or gene products can be modulated and theplants can be tested by screening for the desired trait. Specifically,the gene, mRNA levels, or protein levels can be altered in a plantutilizing the procedures described herein and the phenotypes can bescreened for variants as in Winkler et al. (1998) Plant Physiol 118:743-50 and assayed, for example, in accordance to Alvarez et al., (1998)Cell 92: 773-784; Halhbrock and Scheel, (1989) Ann. Rev. Plant Physiol.Plant Mol. Biol. 40: 347-369; Lamb et al., (1997) Ann. Rev. Plant Mol.Biol. Plant Physiol. 48: 251-275; Lapwood et al. (1984) Plant Pathol.33: 13-20; Levine et al. (1996) Curr. Biol. 6: 427-437; McKersie et al.,(2000) Plant Physiol. 122: 1427-1437; Olson and Varner (1993) Plant J.4: 887-892; Pastore et al., (2000), FEBS Lett 470: 88-92; Pastori etal., (1997) Plant Physiol. 113: 411-418; Romero-Puertas et al., (1999)Free Radic. Res. 1999 31 Suppl: S25-31; Shirataki et al., Anticancer Res20: 423-426 (2000); Wu et al., (1995) Plant Cell 7: 1357-1368.

III.E.12.b. Use of Defense Responsive Genes to Modulate BiochemicalActivities

The activities of one or more of the defense (LOL2) responsive genes canbe modulated to change biochemical or metabolic activities and/orpathways such as those noted below. Such biological activities aredocumented and can be measured according to the citations above andincluded in the Table below:

BIOCHEMICAL OR METABOLIC ACTIVITIES CITATIONS INCLUDING PROCESS AND/ORPATHWAYS ASSAYS Resistance To Pathogens Induction Of ROS Signaling Wuet. al. (1995) Plant Cell 7: Pathways 1357-68 Modulation Of Nitric OxideDelledonne et al. (1998) Nature Signaling 394: 585-588 Induction Of PRProteins, Chamnongpol et. al. (1998) Proc. Phytoalexins, And DefenseNat. Acad Sci USA 12; 95: 5818-23. Pathways Davis et al. (1993)Phytochemistry 32: 607-611 Induction Of Cellular Chen et. al. Plant J.(1996) 10: 955-966 Protectant Genes Such As Gadea et. al. (1999) Mol GenGenet Glutathione S-Transferase 262: 212-219 (GST) And Ascorbate Wuet.al.(1995) Plant Cell 7: Peroxidase 1357-68 ROS Levels FollowingOrozco-Cardenas and Ryan (1999) Wounding And Changes In Proc. Nat. Acad.Sci. USA Physical Pressure 25; 96: 6553-7. Yahraus et al. (1995) PlantPhysiol. 109: 1259-1266 Salicyclic Acid Levels And Durner and Klessig(1996) Signaling J. Biol. Chem. 271: 28492-501 Responses To WoundingExpression Of Genes Involved Legendre et al. (1993) Plant In WoundRepair And Cell Physiol. 102: 233-240 Division Responses To ExpressionOf Genes Involved Shi et al. (2000) Proc. Natl. Acad. EnvironmentalStress In Responses To Drought, Sci. USA 97: 6896-6901 Cold, Salt, HeavyMetals Reinforcement Of Cell Modulation Of The Production Bradley et al.(1992) Cell 70, 21-30 Walls Of ExtracTable Proline-Rich ProteinModulation Of Lignification Mansouri et al. (1999) Physiol. Plant 106:355-362 Programmed Cell Death Induction Of Pcd Activating Levine et al.(1996) Curr. Biol. 6: Genes 427-437. Reynolds et.al. (1998) Biochem. J.330: 115-20 Suppression Of PCD Pennell and Lamb (1997) Plant SuppressingGenes Cell 9, 1157-1168

Other biological activities that can be modulated by the LOL2 responsivegenes and their products are listed in the Reference tables. Assays fordetecting such biological activities are described in the Protein Domaintable.

LOL2 responsive genes are characteristically differentially transcribedin response to fluctuating levels of disease. MA_diff table reports thechanges in transcript levels of various LOL2 responsive genes in thelol-2 line versus control plants.

The data from this experiment reveal a number of types of LOL2responsive genes and gene products. Profiles of individual LOL2responsive genes are shown in the Table below with examples of whichassociated biological activities are modulated when the activities ofone or more such genes vary in plants.

EXAMPLES OF GENE FUNCTIONAL BIOCHEMICAL EXPRESSION CATEGORYPHYSIOLOGICAL ACTIVITY LEVELS OF GENE CONSequence OF GENE PRODUCTSUpregulated Early ROS Perception and Transcription factors, transcriptsResponders to Response kinases, phosphatases, GTP- the LOL2 bindingproteins (G- Mutation proteins), leucine rich repeat proteins (LRRs),transporters, calcium binding proteins, chromatin remodeling proteinsInitiation of Gene Glutathione S-transferase Transcription (GST), heatshock proteins, salicylic acid (SA) response pathway proteins, jasmonateresponse pathway proteins, dehydrins, peroxidases, catalases DelayedInitiation of Defence Proteases, pathogen Responders to GeneTranscription response (PR) proteins, the LOL2 cellulases, chitinases,Mutation cutinases, glucanases, other degrading enzymes, calcium channelblockers, phenylalanine ammonia lyase, proteins of defense pathways,cell wall proteins incuding proline rich proteins and glycine richproteins, epoxide hydrolase, methyl transferases Activation of celldeath Transcription factors pathways kinases, phosphatases, DNAsurveillance proteins, p53, proteases, endonucleases, GTP-bindingproteins (G- proteins), leucine rich repeat proteins (LRRs),transporters, calcium binding proteins, mitochondrial and chloroplastenergy related proteins, ribosome inactivating proteins Initiation ofCellular Reactive oxygen scavenging Protectant Gene enzymes, GST,catalase, Transcription peroxidase, ascorbate oxidase DownregulatedEarly Negative regulation of Transcription factors, transcriptsResponders to LOL2 Mutation kinases, phosphatases, GTP- the LOL2inducible pathways binding proteins (G- Mutation released proteins),leucine rich repeat proteins (LRRs), transporters, calcium bindingproteins, chromatin remodelling proteins Genes Negative regulation ofTranscription factors, Repressed by ROS inducible kinases, phosphatases,GTP- the LOL2 pathways released binding proteins (G- Mutation proteins),leucine rich repeat proteins (LRRs), transporters, calcium bindingproteins, chromatin remodelling proteins Delayed Negative regulation ofTranscription factors, Responders to LOL2 Mutation kinases,phosphatases, GTP- the LOL2 inducible pathways binding proteins (G-Mutation released proteins), leucine rich repeat proteins (LRRs),transporters, calcium binding proteins, chromatin remodelling proteinsGenes Negative Regulation Of Transcription Factors, Repressed By GenesSuppressing Kinases, Phosphatases, The LOL2 Programmed Cell GTP-BindingProteins (G- Mutation Death Released Proteins), Leucine Rich RepeatProteins (Lrrs), Transporters, Calcium Binding Proteins, ChromatinRemodelling Proteins

Use of Promoters of Defense Responsive Genes

Promoters of Defense responsive genes are useful for transcription ofany desired polynucleotide or plant or non-plant origin. Further, anydesired sequence can be transcribed in a similar temporal, tissue, orenvironmentally specific patterns as the Defense responsive genes wherethe desired sequence is operably linked to a promoter of a Defenseresponsive gene. The protein product of such a polynucleotide is usuallysynthesized in the same cells, in response to the same stimuli as theprotein product of the gene from which the promoter was derived. Suchpromoter are also useful to produce antisense mRNAs to down-regulate theproduct of proteins, or to produce sense mRNAs to down-regulate mRNAsvia sense suppression.

III.E.14. Iron Responsive Genes, Gene Components and Products

Iron (Fe) deficiency in humans is the most prevalent nutritional problemworldwide today. Increasing iron availability via diet is a sustainablemalnutrition solution for many of the world's nations. One-third of theworld's soils, however, are iron deficient. Consequently, to form afood-based solution to iron malnutrition, we need a better understandingof iron uptake, storage and utilization by plants. Furthermore, exposureto non-toxic Fe levels appears to affect inherent plant defensemechanisms. Consequently, exploring the effects of Fe exposure haspotential for advances in plant disease resistance in addition to humannutrition.

Microarray technology allows monitoring of gene expression levels forthousands of genes in a single experiment. This is achieved bysimultaneously hybridizing two differentially labeled fluorescent FeNApools to glass slides that contain spots of DNA (Schena et al. (1995)Science 270: 467-70). The Arabidopsis Functional Genomics Consortium(AFGC) has recently made public the results from such microarrayexperiments conducted with AFGC chips containing 10,000 non-redundantESTs, selected from 37,000 randomly sequenced ESTs generated from mRNAof different tissues and developmental stages.

The sequences of the ESTs showing at least two-fold increases ordecreases over the controls were identified, compared to the Ceres fulllength FeNA and genomic sequence databanks, and identical Ceres clonesidentified. MA_diff table reports the results of this analysis,indicating those Ceres clones that are up or down regulated overcontrols, thereby indicating the Ceres clones which are iron responsivegenes.

The MA_diff Table(s) reports the transcript levels of the experiment(see EXPT ID: Iron (relating to SMD 7114, SMD 7115, SMD 7125)). Fortranscripts that had higher levels in the samples than the control, a“+” is shown. A “−” is shown for when transcript levels were reduced inroot tips as compared to the control. For more experimental detail seethe Example section below.

Iron genes are those sequences that showed differential expression ascompared to controls, namely those sequences identified in the MA_difftables with a “+” or “−” indication.

Iron Genes Identified by Cluster Analyses of Differential Expression

Iron Genes Identified by Correlation to Genes that are DifferentiallyExpressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of Iron genes is any group in the MA_clust thatcomprises a cDNA ID that also appears in Expt ID Iron (relating to SMD7114, SMD 7115, SMD 7125) of the MA_diff table(s).

Iron Genes Identified by Correlation to Genes that Cause PhysiologicalConsequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of Irongenes. A group in the MA_clust is considered a Iron pathway or networkif the group comprises a cDNA ID that also appears in Knock-in orKnock-out tables that causes one or more of the phenotypes described insection above.

Iron Genes Identified by Amino Acid Sequence Similarity

Iron genes from other plant species typically encode polypeptides thatshare amino acid similarity to the sequences encoded by corn andArabidopsis Iron genes. Groups of Iron genes are identified in theProtein Group table. In this table, any protein group that comprises apeptide ID that corresponds to a cDNA ID member of a Iron pathway ornetwork is a group of proteins that also exhibits Ironfunctions/utilities.

III.E.14.a. Use of Iron Responsive Genes to Modulate Phenotypes

Iron responsive genes and gene products are useful to or modulate one ormore phenotypes including growth; roots; root hair formation; stems,leaves; development; senescence; plant nutrition; uptake andassimilation of organic compounds; uptake and assimilation of inorganiccompounds; animal (including human) nutrition; improved dietary mineralnutrition; stress response metabolic detoxification; and heavy metals.

To improve any of the phenotype(s) above, activities of one or more ofthe iron responsive genes or gene products can be modulated and testedby screening for the desired trait. Specifically, the gene, mRNA levels,or protein levels can be altered in a plant utilizing the proceduresdescribed herein and the phenotypes can be assayed. As an example, aplant can be transformed according to Bechtold and Pelletier (1998,Methods. Mol. Biol. 82:259-266) and visually inspected for the desiredphenotype or metabolically and/or functionally assayed according toSchmidt et al. (2000, Plant Physiol 122:1109-18), Meagher (2000) CurrentOpinion in Plant Biology 3: 153-62), Deak (1999, Nature Biotechnology(1999, Nature Biotechnology 17: 192-96), Wei and Theil (2000, J. BiolChem 275: 17488-93) and Vansuyt et al. (1997, FEBS Letters 410:195-200).

III.E.14.b. Use of Iron-Responsive Genes, Gene Components and Productsto Modulate Biochemical Activities

The activities of one or more of the iron responsive genes can bemodulated to change biochemical or metabolic activities and/or pathwayssuch as those noted below. Such biological activities can be measuredaccording to the citations included in the Table below:

BIOCHEMICAL OR CITATIONS METABOLIC ACTIVITIES INCLUDING PROCESS AND/ORPATHWAYS ASSAYS Growth, Root Growth Robinson et al. (1999)Differentiation Initiation of root Nature 397: 694-97 and hairsDevelopment Metabolisms Iron sensing Thomine et al. (2000) Iron uptakeand transport PNAS USA 97: 4991-6 decreased iron Thomine et al. (2000)transport PNAS USA 97: 4991-6 Zhu (1999) Plant phytoremediation Physiol119: 73-79 Plant Protection from oxidative Deak (1999) Nature Defensesdamage Biotechnology 17: 192-6 Signaling Specific gene Brand andPerrimon transcription gene (1993) Development silencing 118: 401-415

Other biological activities that can be modulated by the iron responsivegenes and gene products are listed in the REFERENCE Table. Assays fordetecting such biological activities are described in the Protein Domaintable.

Iron responsive genes are characteristically differentially transcribedin response to fluctuating iron levels or concentrations, whetherinternal or external to an organism or cell. MA_diff table reports thechanges in transcript levels of various iron responsive genes.

The microarray comparison consists of probes prepared from root RNA ofA. thaliana (Columbia) seedlings grown under iron-sufficient conditionsand seedlings grown under iron-deficient. The data from this experimentreveal a number of types genes and gene products. Profiles of thesedifferent iron responsive genes are shown in the Table below withexamples of associated biological activities.

EXAMPLES OF TRANSCRIPT PHYSIOLOGICAL BIOCHEMICAL LEVELS TYPE OF GENESCONSEQUENCES ACTIVITY Up regulated responders to iron Iron perceptionTransporters transcripts application Iron uptake and Metabolic enzymestransport Change in cell Iron metabolism membrane structure Synthesis ofand potential secondary Kinases and metabolites phosphatases and/orproteins Transcription Modulation of activators iron response Change inchromatin transduction structure and/or pathways localized DNA Specificgene topology transcription initiation Down-regulated responder to ironNegative Transcription factors transcripts repressors of iron stateregulation of iron Change in protein of metabolism pathways structure byGenes with Changes in phosphorylation discontinued pathways and(kinases) or expression or processes dephosphoryaltion unsTable mRNA inoperating in cells (phosphatases) presence of iron Changes in otherChange in chromatin metabolisms than structure and/or iron DNA topologyStability of factors for protein synthesis and degradation Metabolicenzymes

Use of Promoters of Iron Responsive Genes

Promoters of Iron responsive genes are useful for transcription of anydesired polynucleotide or plant or non-plant origin. Further, anydesired sequence can be transcribed in a similar temporal, tissue, orenvironmentally specific patterns as the Iron responsive genes where thedesired sequence is operably linked to a promoter of a Iron responsivegene. The protein product of such a polynucleotide is usuallysynthesized in the same cells, in response to the same stimuli as theprotein product of the gene from which the promoter was derived. Suchpromoter are also useful to produce antisense mRNAs to down-regulate theproduct of proteins, or to produce sense mRNAs to down-regulate mRNAsvia sense suppression.

III.E.15. Shade Responsive Genes, Gene Components and Products

Plants sense the ratio of Red (R):Far Red (FR) light in theirenvironment and respond differently to particular ratios. A low R:FRratio, for example, enhances cell elongation and favors flowering overleaf production. The changes in R:FR ratios mimic and cause the shadingresponse effects in plants. The response of a plant to shade in thecanopy structures of agricultural crop fields influences crop yieldssignificantly. Therefore manipulation of genes regulating the shadeavoidance responses can improve crop yields. While phytochromes mediatethe shade avoidance response, the down-stream factors participating inthis pathway are largely unknown. One potential downstream participant,ATHB-2, is a member of the HD-Zip class of transcription factors andshows a strong and rapid response to changes in the R:FR ratio. ATHB-2overexpressors have a thinner root mass, smaller and fewer leaves andlonger hypocotyls and petioles. This elongation arises from longerepidermal and cortical cells, and a decrease in secondary vasculartissues, paralleling the changes observed in wild-type seedlings grownunder conditions simulating canopy shade. On the other hand, plants withreduced ATHB-2 expression have a thick root mass and many larger leavesand shorter hypocotyls and petioles. Here, the changes in the hypocotylresult from shorter epidermal and cortical cells and increasedproliferation of vascular tissue. Interestingly, application of Auxin isable to reverse the root phenotypic consequences of high ATHB-2 levels,restoring the wild-type phenotype. Consequently, given that ATHB-2 istightly regulated by phytochrome, these data suggest that ATHB-2 maylink the Auxin and phytochrome pathways in the shade avoidance responsepathway.

Changes in R:FR ratios promote changes in gene expression. Microarraytechnology allows monitoring of gene expression levels for thousands ofgenes in a single experiment. This is achieved by hybridizing labeledfluorescent cDNA pools to glass slides that contain spots of DNA (Schenaet al. (1995) Science 270: 467-70). The US Arabidopsis FunctionalGenomics Consortium (AFGC) has recently made public the results fromsuch microarray experiments conducted with AFGC chips containing about10,000 non-redundant ESTs, selected from about 37,000 randomly sequencedESTs generated from mRNA of different tissues and developmental stages.

The sequences of the ESTs showing at least two-fold increases ordecreases in plants given 4 hours of FR rich light after growth in highR:FR light compared with the controls of plants grown in high R:FR lightonly, were identified, compared to the Ceres full length cDNA andgenomic sequence databanks, and equivalent Ceres clones identified. TheMA_diff table(s) report(s) the results of this analysis, indicatingthose Ceres clones which are up or down regulated over controls, therebyindicating the Ceres clones which are shade avoidance responsive genes.

Examples of far red light induced, shade avoidance responsive genes andgene products are shown in the Reference and Sequence Tables. Thesegenes and/or products are responsible for effects on traits such asplant vigor and seed yield.

While far red light, shade avoidance responsive polynucleotides and geneproducts can act alone, combinations of these polynucleotides alsoaffect growth and development. Useful combinations include differentshade avoidance responsive polynucleotides and/or gene products thathave similar transcription profiles or similar biological activities,and members of the same or similar biochemical pathways. In addition,the combination of a shade avoidance responsive polynucleotide and/orgene product with another environmentally responsive polynucleotides isalso useful because of the interactions that exist betweenhormone-regulated pathways, stress and pathogen induced pathways,nutritional pathways, light induced pathways and development. Here, inaddition to polynucleotides having similar transcription profiles and/orbiological activities, useful combinations include polynucleotides thatmay have different transcription profiles but which participate incommon or overlapping pathways.

Such far red light induced shade avoidance responsive genes and geneproducts can function to either increase or dampen the above phenotypesor activities either in response to changes in far red light or in theabsence of far red light fluctuations. The MA_diff Table(s) reports thetranscript levels of the experiment (see EXPT ID: Shade (relating to SMD8130, SMD 7230)). For transcripts that had higher levels in the samplesthan the control, a “+” is shown. A “−” is shown for when transcriptlevels were reduced in root tips as compared to the control. For moreexperimental detail see the Example section below.

Shade genes are those sequences that showed differential expression ascompared to controls, namely those sequences identified in the MA_difftables with a “+” or “−” indication.

Shade Genes Identified by Cluster Analyses of Differential Expression

Shade Genes Identified by Correlation to Genes that are DifferentiallyExpressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of Shade genes is any group in the MA_clust thatcomprises a cDNA ID that also appears in Expt ID Shade (relating to SMD8130, SMD 7230) of the MA_diff table(s).

Shade Genes Identified by Correlation to Genes that Cause PhysiologicalConsequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of Shadegenes. A group in the MA_clust is considered a Shade pathway or networkif the group comprises a cDNA ID that also appears in Knock-in orKnock-out tables that causes one or more of the phenotypes described insection above.

Shade Genes Identified by Amino Acid Sequence Similarity

Shade genes from other plant species typically encode polypeptides thatshare amino acid similarity to the sequences encoded by corn andArabidopsis Shade genes. Groups of Shade genes are identified in theProtein Group table. In this table, any protein group that comprises apeptide ID that corresponds to a cDNA ID member of a Shade pathway ornetwork is a group of proteins that also exhibits Shadefunctions/utilities.

Further, promoters of shade avoidance responsive genes, as described inthe Reference tables, for example, are useful to modulate transcriptionthat is induced by shade avoidance or any of the following phenotypes orbiological activities below. Further, any desired sequence can betranscribed in similar temporal, tissue, or environmentally specificpatterns as the shade avoidance responsive genes when the desiredsequence is operably linked to a promoter of a circadian (clock)responsive gene.

III.E.15.a. Use of Far Red Responsive, Shade Avoidance Response Genes toModulate Phenotypes

High FR:R, shade avoidance responsive genes and gene products can beused to alter or modulate one or more phenotypes including growth;roots; elongation; lateral root formation; stems; elongation; expansion;leaves; expansion; carotenoid composition; development; cell;photosynthetic apparatus; efficiency; flower; flowering time; fruit;seed; dormancy; control rate and timing of germination; prolongs seedstorage and viability; inhibition of hydrolytic enzyme synthesis; seedand fruit yield; senescence; abscission; leaf fall; flower longevity;differentiation; vascularization; and shade (avoidance) responses inplant architecture.

To regulate any of the phenotype(s) above, activities of one or more ofthe High FR:R light, shade avoidance responsive genes or gene productscan be modulated and the plants tested by screening for the desiredtrait. Specifically, the gene, mRNA levels, or protein levels can bealtered in a plant utilizing the procedures described herein and thephenotypes can be assayed. As an example, a plant can be transformedaccording to Bechtold and Pelletier (1998, Methods. Mol. Biol.82:259-266) and/or screened for variants as in Winkler et al. (1998)Plant Physiol 118: 743-50 and visually inspected for the desiredphenotype or metabolically and/or functionally assayed according toCarabelli et al. (1996, PNAS USA 93: 3530-3535), Aguirrezabal andTardieu (1996, J Exp Bot 47: 411-20), Heyer et al. (1995, Plant Physiol109: 53-61), Garcia-Plazaola et al. (1997, J Exp Bot 48: 1667-74),Schwanz et al. (1996, J Exp Bot 47L 1941-50), Koehne et al. (1999,Biochem Biophys Acta 1412:94-107), Melis (1984, J Cell Biochem 24:271-85), Steindeler et al. (1999, Development 126: 4235-45), Cruz (1997,J Exp Bot 48: 15-24), Stephanou and Manetas (1997, J Exp Bot 48:1977-85), Grammatikopoulos et al (1999, J Exp Bot 50:517-21), Krause etal. (1999, Plant Physiol 121: 1349-58), Aukerman et al. (1997, PlantCell 9: 1317-26), Wagner et al. (1997, Plant Cell 9: 731-43), Weinig(2000) Evolution Int J Org Evolution 54: 124-26), Cocburn et al. (1996,J Exp Bot 47: 647-53), Devlin et al. (1999, Plant Physiol 119: 909-15),Devlin et al. (1998, Plant Cell 10: 1479-87), Finlayson et al. (1998,Plant Physiol 116: 17-25), Morelli and Ruberti (2000, Plant Physiol 122:621-26), Aphalo et al. (1999, J Exp Bot 50: 1629-34), Sims et al. (1999,J Exp Bot 50:50:645-53) and Ballare (1999, Trends Plant Sci 4: 97-102).

III.E.15.b. Use of Far Red Light, Shade Avoidance Responsive Genes toModulate Biochemical Activities

The activities of one or more of the far red light, shade avoidanceresponsive genes can be modulated to change biochemical or metabolicactivities and/or pathways such as those noted below. Such biologicalactivities can be measured according to the citations included in theTable below:

BIOCHEMICAL OR METABOLIC ACTIVITIES AND/OR CITATIONS INCLUDING PROCESSPATHWAYS ASSAYS Cell Growth and Cell Elongation Carabelli et al. (1996)PNAS USA Differentiation 93: 3530-35 Leaf Expansion Heyer et al. (1995)Plant Physiol 109: 53-61 Photosynthesis Development of Jagtap et al.(1998) J Exp Bot 49: Photosynthetic Apparatus 1715-21 Melis (1984) JCell Biochem 24: 271-285 McCain (1995) Biophys J 69: 1105-10 CarotenoidComposition Garcia-Plazaola et al (1997) J Exp Bot 48: 1667-74Carbon/Nitrogen Carbon and Nitrogen Cruz (1997) J Exp Bot 48: 15-24Metabolism Assimilation Far red light, shade Newton AL, Sharpe BK, KwanA, avoidance response Mackay JP, Crossley M. J Biol binding bytranscription Chem. 2000May19; 275(20): 15128-34; factors Lopez RiberaI, Ruiz-Avila L, Puigdomenech P. Biochem Biophys Res Commun. 1997 Jul18; 236(2): 510-6; de Pater S, Greco V, Pham K, Memelink J, Kijne J.Nucleic Acids Res. 1996 Dec 1; 24(23): 4624-31. Signaling UV LightPerception Stephanou and Manetas (1997) J Exp Bot 48: 1977-85Far-red/Red Light Aukerman et al. (1997) Plant Cell Perception 9:1317-26 Wagner et al. (1997) Plant Cell 9: 731-43 Interaction of “ShadeFinlayson et al. (1998) Plant Factor” with Ethylene Physiol 116: 17-25Production/Transduction Interaction of “Shade Reed et al. (1998) PlantPhysiol Factor” with Auxin 118: 1369-78 Production/Transduction Plant toPlant signalling Sims et al. (1999) J Exp Bot 50: 645-53

Other biological activities that can be modulated by shade avoidanceresponse genes and their products are listed in the REF TABLES. Assaysfor detecting such biological activities are described in the ProteinDomain table.

High FR:R, shade avoidance responsive genes are differentiallytranscribed in response to high FR:R ratios. The microarray comparisonto reveal such genes consisted of probes prepared from RNA isolated fromthe aerial tissues of A. thaliana (Columbia) two-week old seedlingsgrown in high R:FR ratios compared to seedlings grown in high R:FRratios followed by 4 hours of FR-rich light treatment. The data fromthis experiment reveal a number of types genes and gene products andexamples of the classes of genes are given in the Table below.

EXAMPLES OF TRANSCRIPT PHYSIOLOGICAL BIOCHEMICAL LEVELS TYPE OF GENESCONSEQUENCES ACTIVITY Up regulated Responders to high Far red lightTransporters transcripts FR:R light ratios perception Metabolic enzymesGenes induced by Metabolism Change in cell high FR:R light ratioaffected by far red membrane structure light and potential Synthesis ofKinases and secondary phosphatases metabolites and/or Transcriptionproteins activators Modulation of Change in chromatin high FR:R lightstructure and/or ratio transduction localized DNA pathways topologySpecific gene Leaf production transcription factors initiationDown-regulated Responders to high Changes in Transcription factorstranscripts FR:R light ratios pathways and Change in protein Genesrepressed by processes structure by high FR:R light ratio operating incells phosphorylation Genes with Changes in (kinases) or discontinuedmetabolisms other dephosphorylation expression or than far red(phosphatases) unsTable mRNA stimulated Change in chromatin during highFR:R pathways structure and/or DNA ratio light topology Stability offactors for protein synthesis and degradation Metabolic enzymes Cellelongation factors Flowering promotion factors

Use of Promoters of Shade Avoidance Genes

Promoters of Shade Avoidance genes are useful for transcription of anydesired polynucleotide or plant or non-plant origin. Further, anydesired sequence can be transcribed in a similar temporal, tissue, orenvironmentally specific patterns as the Shade Avoidance genes where thedesired sequence is operably linked to a promoter of a Shade Avoidancegene. The protein product of such a polynucleotide is usuallysynthesized in the same cells, in response to the same stimuli as theprotein product of the gene from which the promoter was derived. Suchpromoter are also useful to produce antisense mRNAs to down-regulate theproduct of proteins, or to produce sense mRNAs to down-regulate mRNAsvia sense suppression.

III.E.16. Sulfur Responsive Genes, Gene Components and Products

Sulfur is one of the important macronutrients required by plants. It istaken up from the soil solution by roots as in the form of sulfate anionwhich higher plants are dependent on to fulfill their nutritional sulfurrequirement. After uptake from the soil, sulfate is either accumulatedand stored in vacuole or it is assimilated into various organiccompounds, e.g. cysteine, glutathione, methionine, etc. Thus, plantsalso serve as nutritional sulfur sources for animals. Sulfur can beassimilated in one of two ways: it is either incorporated as sulfate ina reaction called sulfation, or it is first reduced to sulfide, thesubstrate for cysteine synthesis. In plants, majority of sulfur isassimilated in reduced form.

Sulfur comprises a small by vital fraction of the atoms in many proteinmolecules. As disulfide bridges, the sulfur atoms aid in stabilizing thefolded proteins, such cysteine residues. Cys is the firstsulfur-containing amino acids, which in proteins form disulfide bondsthat may affect the tertiary structures and enzyme activities. Thisredox balance is mediated by the disulfide/thiol interchange ofthioredoxin or glutaredoxin using NADPH as an electron donor. Sulfur canalso become sulfhydryl (SH) groups participating in the active sites ofsome enzymes and some enzymes require the aid of small molecules thatcontain sulfur. In addition, the machinery of photosynthesis includessome sulfur-containing compounds, such as ferrodoxin. Thus, sulfateassimilation plays important roles not only in the sulfur nutrition butalso in the ubiquitous process that may regulate the biochemicalreactions of various metabolic pathways.

Deficiency of sulfur leads to a marked chlorosis in younger leaves,which may become white in color. Other symptoms of sulfur deficiencyalso include weak stems and reduced growth. Adding sulfur fertilizer toplants can increase root development and a deeper green color of theleaves in sulfur-deficient plants. However, Sulfur is generallysufficient in soils for two reasons: it is a contaminant in potassiumand other fertilizers and a product of industrial combustion. Sulfurlimitation in plants is thus likely due to the limitation of the uptakeand distribution of sulfate in plants. Seven cell type specific sulfatetransporter genes have been isolated from Arabidopsis. Insulfate-starved plants, expression of the high-affinity transporter,AtST1-1, is induced in root epidermis and cortex for acquisition ofsulfur. The low affinity transporter, AtST2-1 (AST68), accumulates inthe root vascular tissue by sulfate starvation for root-to-shoottransport of sulfate. These studies have shown that the whole-plantprocess of sulfate transport is coordinately regulated by the expressionof these 2 sulfate transporter genes under sulfur limited conditions.Recent studies have proposed that feeding of O-acetylserine, GSH andselenate may regulate the expression of AtST1-1 and AtST2-1 (AST68) inroots either positively or negatively. However, regulatory proteins thatmay directly control the expression of these genes have not beenidentified yet.

It has been established that there are regulatory interactions betweenassimilatory sulfate and nitrate reduction in plants. The twoassimilatory pathways are very similar and well coordinated; deficiencyfor one element was shown to repress the other pathway. The coordinationbetween them should be taken into consideration when one tries to alterone of pathways.

Microarray technology allows monitoring of gene expression levels forthousands of genes in a single experiment. This is achieved bysimultaneously hybridizing two differentially labeled fluorescent cDNApools to glass slides that contain spots of DNA (Schena et al. (1995)Science 270: 467-70). The Arabidopsis Functional Genomics Consortium(AFGC) has recently made public the results from such microarrayexperiments conducted with AFGC chips containing 10,000 non-redundantESTs, selected from 37,000 randomly sequenced ESTs generated from mRNAof different tissues and developmental stages.

The sequences of the ESTs showing at least two-fold increases ordecreases over the controls were identified, compared to the Ceresfull-length cDNA and genomic sequence databanks, and identical Ceresclones identified. MA_diff table reports the results of this analysis,indicating those Ceres clones which are up or down regulated overcontrols, thereby indicating the Ceres clones which are sulfur responseresponsive genes.

The MA_diff Table(s) reports the transcript levels of the experiment(see EXPT ID: Sulfur (relating to SMD 8034, SMD 8035)). For transcriptsthat had higher levels in the samples than the control, a “+” is shown.A “−” is shown for when transcript levels were reduced in root tips ascompared to the control. For more experimental detail see the Examplesection below.

Sulfur genes are those sequences that showed differential expression ascompared to controls, namely those sequences identified in the MA_difftables with a “+” or “−” indication.

Sulfur Genes Identified by Cluster Analyses of Differential Expression

Sulfur Genes Identified by Correlation to Genes that are DifferentiallyExpressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of Sulfur genes is any group in the MA_clust thatcomprises a cDNA ID that also appears in Expt ID Sulfur (relating to SMD8034, SMD 8035) of the MA_diff table(s).

Sulfur Genes Identified by Correlation to Genes that Cause PhysiologicalConsequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of Sulfurgenes. A group in the MA_clust is considered a Sulfur pathway or networkif the group comprises a cDNA ID that also appears in Knock-in orKnock-out tables that causes one or more of the phenotypes described insection above.

Sulfur Genes Identified by Amino Acid Sequence Similarity

Sulfur genes from other plant species typically encode polypeptides thatshare amino acid similarity to the sequences encoded by corn andArabidopsis Sulfur genes. Groups of Sulfur genes are identified in theProtein Group table. In this table, any protein group that comprises apeptide ID that corresponds to a cDNA ID member of a Sulfur pathway ornetwork is a group of proteins that also exhibits Sulfurfunctions/utilities.

III.E.16.a. Use of Sulfur Responsive Genes to Modulate Phenotypes

Sulfur responsive genes and gene products are useful to or modulate oneor more phenotypes including growth; roots; stems; leaves; development;chloroplasts and mitochondria; fruit development; seed development; seedstorage proteins; senescence; differentiation; plastid/chloroplast andmitochondria differentiation; protection against oxidative damage;regulation of enzymes via redox control by thiol groups; metabolicdetoxification; photosynthesis; and carbon dioxide fixation.

To improve any of the phenotype(s) above, activities of one or more ofthe sulfur responsive genes or gene products can be modulated and testedby screening for the desired trait. Specifically, the gene, mRNA levels,or protein levels can be altered in a plant utilizing the proceduresdescribed herein and the phenotypes can be assayed. As an example, aplant can be transformed according to Bechtold and Pelletier (1998,Methods. Mol. Biol. 82:259-266) and visually inspected for the desiredphenotype or metabolically and/or functionally assayed according toSaito et al. (1994, Plant Physiol. 106: 887-95), Takahashi et al (1997,Proc. Natl. Acad. Sci. USA 94: 11102-07) and Koprivova et al. (2000,Plant Physiol. 122: 737-46).

III.E. 16.b. Use of Sulfur-Responsive Genes, Gene Components andProducts to Modulate Biochemical Activities

The activities of one or more of the sulfur responsive genes can bemodulated to change biochemical or metabolic activities and/or pathwayssuch as those noted below. Such biological activities can be measuredaccording to the citations included in the Table below:

BIOCHEMICAL OR METABOLIC ACTIVITIES CITATIONS INCLUDING PROCESS AND/ORPATHWAYS ASSAYS Growth, Root Klein and Klein (1988) MineralDifferentiation and Leaf Nutrition, In CM Wilson and J Development StemGregory, eds Fundamentals of Chloroplast/Mitochondria Plant Science.Harper and Row development/differentiation Publishers, Inc., NY, p163Rost et al. (1984) The Absorption and Transport System, In R Bem, ed,Botany-A Brief Introduction to Plant Biology. John Wiley and Sons, NY,p96. Huluigue et al. (2000) Biochem Biophys Res Commun 271: 380-5Kapazoglou et al. (2000) Eur J Biochem 267: 352-60 Seed storage proteinKim et al. (1999) 209: 282-9 synthesis Metabolisms Sulfate uptake andtransport Takahashi et al. (1997) Proc Natl Acad Sci USA 94: 11102-07Cysteine Biosynthesis Saito et al. (1992) Proc Natl Acad Sci USA 89:8078-82 Hesse et al. (1999) Amino Acids 16: 113-31 Methioninebiosynthesis Bourgis et al. (1999) Plant Cell 11: 1485-98 Carbon dioxidefixation in Buchana (1991) Arch Biochem photosynthesis Biophys 288: 1-9Thioredoxin reduction Leustek and Saito (1999) Plant Phyiol 120: 637-43Mamedova et al. (1999) FEBS Lett 462: 421-4 Nitrogen metabolismKoprivova et al. (2000) Plant Physiol. 122: 737-46 Yamaguchi et al.(1999) Biosci Biotechnol Biochem 63: 762-6 Plant Defenses Reduction ofoxidative May et al. (1998) J Expt Bio 49: stress - oxygen metabolism649-67 and reactive oxygen species Kreuz et al. (1996) Plant PhysiolDetoxification of toxins, 111: 349-53 xenobiotics and heavy Zhao et al.(1998) Plant Cell 10: metals 359-70 Defense against pathogens Kyung andFleming (1997) J Food or microbes Prot 60: 67-71 Disease prevention byFahey et al. (1997) Proc Natl Acad secondary sulfur-containing Sci USA94: 10367-72 compounds Activation of kinases and Davis et al. (1999)Plant Cell 11: phosphatases 1179-90

Other biological activities that can be modulated by the sulfurresponsive genes and gene products are listed in the REFERENCE Table.Assays for detecting such biological activities are described in theProtein Domain table.

Sulfur responsive genes are characteristically differentiallytranscribed in response to fluctuating sulfur levels or concentrations,whether internal or external to an organism or cell. MA_diff tablereports the changes in transcript levels of various sulfur responsivegenes.

Profiles of these different sulfur responsive genes are shown in theTable below with examples of associated biological activities.

EXAMPLES OF TRANSCRIPT PHYSIOLOGICAL BIOCHEMICAL LEVELS TYPE OF GENESCONSEQUENCES ACTIVITY Up regulated Responders to sulfur Sulfurperception Transporters transcripts Application Sulfur uptake andMetabolic enzymes transport Change in cell Sulfur metabolism membranestructure Synthesis of and potential secondary Kinases and metabolitesand/or phosphatases proteins Transcription Modulation of activatorssulfur response Change in chromatin transduction structure and/orpathways localized DNA Specific gene topology transcription Redoxcontrol initiation Down-regulated responder to sulfur NegativeTranscription factors transcripts repressors of sulfur regulation ofChange in protein state of metabolism sulfur pathways structure by Geneswith Changes in phosphorylation discontinued pathways and (kinases) orexpression processes dephosphoryaltion or unsTable mRNA in operating incells (phosphatases) presence of sulfur Changes in other Change inchromatin metabolisms than structure and/or DNA sulfur topologyStability of factors for protein synthesis and degradation Metabolicenzymes

Use of Promoters of Sulfur Responsive Genes

Promoters of Sulfur responsive genes are useful for transcription of anydesired polynucleotide or plant or non-plant origin. Further, anydesired sequence can be transcribed in a similar temporal, tissue, orenvironmentally specific patterns as the Sulfur responsive genes wherethe desired sequence is operably linked to a promoter of a Sulfurresponsive gene. The protein product of such a polynucleotide is usuallysynthesized in the same cells, in response to the same stimuli as theprotein product of the gene from which the promoter was derived. Suchpromoter are also useful to produce antisense mRNAs to down-regulate theproduct of proteins, or to produce sense mRNAs to down-regulate mRNAsvia sense suppression.

III.E.17. Zinc Responsive Genes, Gene Components and Products

Phytoremediation of soils contaminated with toxic levels of heavy metalsrequires the understanding of plant metal transport and tolerance. Thenumerous Arabidopsis thaliana studies have given scientists thepotential for dissection and elucidation of plant micronutrient/heavymetal uptake and accumulation pathways. It has been shown alteredregulation of ZNT1, a Zn/Cd transporter, contributes to high Zn uptake.Isolation and characterization of Zn/Cd hyperaccumulation genes mayallow expression in higher biomass plant species for efficientcontaminated soil clean up. Identification of additional Zn transport,tolerance and nutrition-related genes involved in heavy metalaccumulation will enable manipulation of increased uptake (forphytoremediation) as well as limitation of uptake or leak pathways thatcontribute to toxicity in crop plants. Additionally, Zn-binding ligandsinvolved in Zn homeostasis or tolerance may be identified, as well asfactors affecting the activity or expression of Zn binding transcriptionfactors. Gene products acting in concert to effect Zn uptake, whichwould not have been identified in complementation experiments, includingmultimeric transporter proteins, could also be identified.

Microarray technology allows monitoring of gene expression levels forthousands of genes in a single experiment. This is achieved bysimultaneously hybridizing two differentially labeled fluorescent cDNApools to glass slides that contain spots of DNA (Schena et al. (1995)Science 270: 467-70). The Arabidopsis Functional Genomics Consortium(AFGC) has recently made public the results from such microarrayexperiments conducted with AFGC chips containing 10,000 non-redundantESTs, selected from 37,000 randomly sequenced ESTs generated from mRNAof different tissues and developmental stages.

The sequences of the ESTs showing at least two-fold increases ordecreases over the controls were identified, compared to the Ceresfull-length cDNA and genomic sequence databanks, and identical Ceresclones identified. The Zn response information was then used inconjunction with the existing annotation to attribute biologicalfunction or utility to the full-length cDNA and corresponding genomicsequence.

The MA_diff Table(s) reports the transcript levels of the experiment(see EXPT ID: Zinc (relating to SMD 7310, SMD 7311)). For transcriptsthat had higher levels in the samples than the control, a “+” is shown.A “−” is shown for when transcript levels were reduced in root tips ascompared to the control. For more experimental detail see the Examplesection below.

Zinc genes are those sequences that showed differential expression ascompared to controls, namely those sequences identified in the MA_difftables with a “+” or “−” indication.

Zinc Genes Identified by Cluster Analyses of Differential Expression

Zinc Genes Identified by Correlation to Genes that are DifferentiallyExpressed

As described above, the transcription profiles of genes that acttogether are well correlated. Applicants not only have identified thegenes that are differentially expressed in the microarray experiments,but also have identified the genes that act in concert with them. TheMA_clust table indicates groups of genes that have well correlatedtranscription profiles and therefore participate in the same pathway ornetwork.

A pathway or network of Zinc genes is any group in the MA_clust thatcomprises a cDNA ID that also appears in Expt ID Zinc (relating to SMD7310, SMD 7311) of the MA_diff table(s).

Zinc Genes Identified by Correlation to Genes that Cause PhysiologicalConsequences

Additionally, the differential expression data and the phenotypicobservations can be merged to identify pathways or networks of Zincgenes. A group in the MA_clust is considered a Zinc pathway or networkif the group comprises a cDNA ID that also appears in Knock-in orKnock-out tables that causes one or more of the phenotypes described insection above.

Zinc Genes Identified by Amino Acid Sequence Similarity

Zinc genes from other plant species typically encode polypeptides thatshare amino acid similarity to the sequences encoded by corn andArabidopsis Zinc genes. Groups of Zinc genes are identified in theProtein Group table. In this table, any protein group that comprises apeptide ID that corresponds to a cDNA ID member of a Zinc pathway ornetwork is a group of proteins that also exhibits Zincfunctions/utilities.

III.E.17.a. Use of Zn Transport, Tolerance and Nutrition-Related Genesto Modulate Phenotypes

Changes in zinc concentration in the surrounding environment or incontact with a plant results in modulation of many genes and geneproducts. Examples of such zinc responsive genes and gene products areshown in the Reference, Sequence tables, Protein Group, Protein GroupMatrix, MA_diff, and MA_clust tables. These genes and/or products areresponsible for effects on traits such as plant vigor and seed yield.

While zinc responsive polynucleotides and gene products can act alone,combinations of these polynucleotides also affect growth anddevelopment. Useful combinations include different zinc responsivepolynucleotides and/or gene products that have similar transcriptionprofiles or similar biological activities, and members of the same orsimilar biochemical pathways. In addition, the combination of a zincresponsive polynucleotide and/or gene product with anotherenvironmentally responsive polynucleotide is also useful because of theinteractions that exist between hormone-regulated pathways, stresspathways, nutritional pathways and development. Here, in addition topolynucleotides having similar transcription profiles and/or biologicalactivities, useful combinations include polynucleotides that may havedifferent transcription profiles but which participate in a commonpathway.

Such zinc responsive genes and gene products can function to eitherincrease or dampen the above phenotypes or activities either

-   -   in response to changes in zinc concentration or    -   in the absence of zinc fluctuations.

Zn transport, tolerance and nutrition-related genes and gene productscan be used to alter or modulate one or more phenotypes including Znuptake; transport of Zn or other heavy metals into roots;epidermal/cortical uptake; Xylem loading; Zn compartmentation; Xylemunloading; Phloem loading; efflux from cells to apoplast; sequestrationin vacuoles/subcellular compartments; Zn tolerance; chelation of Zn;transport of Zn; metabolic and transcriptional control; activity of Znbinding enzymes; and activity of Zn binding transcription factors.

To improve any of the phenotype(s) above, activities of one or more ofthe Zn transport, tolerance and nutrition-related genes or gene productscan be modulated and the plants can be tested by screening for thedesired trait. Specifically, the gene, mRNA levels, or protein levelscan be altered in a plant utilizing the procedures described herein andthe phenotypes can be assayed, for example, in accordance to Lasat M M,Pence N S, Garvin D F, Ebbs S D, Kochian L V. J Exp Bot. 2000 January;51(342):71-9; Grotz N, Fox T, Connolly E, Park W, Guerinot M L, Eide D.Proc Natl Acad Sci USA. 1998 Jun. 9; 95(12):7220-4; Crowder M W, Maiti MK, Banovic L, Makaroff C A. FEBS Lett. 1997 Dec. 1; 418(3):351-4; Hart JJ, Norvell W A, Welch R M, Sullivan L A, Kochian L V. Plant Physiol.1998 September; 118(1):219-26.

III.E.17.b. Use of Zn Transport, Tolerance and Nutrition-Related Genesto Modulate Biochemical Activities

Alternatively, the activities of one or more of the zinc responsivegenes can be modulated to change biochemical or metabolic activitiesand/or pathways such as those noted below. Such biological activitiescan be measured according to the citations included in the Table below:

BIOCHEMICAL OR METABOLIC ACTIVITIES AND/OR CITATIONS INCLUDING PROCESSPATHWAYS ASSAYS Zn Uptake and Zn Influx Lasat MM, Pence NS, Garvin DF,Assimilation Ebbs SD, Kochian LV. J Exp Bot. 2000 January; 51(342):71-9. Zn compartmentation Hart JJ, Norvell WA, Welch RM, Sullivan LA,Kochian LV. Plant Physiol. 1998 September; 118(1): 219-26. Zn binding bymetabolic Crowder MW, Maiti MK, Banovic L, enzymes Makaroff CA. FEBSLett. 1997 Dec 1; 418(3): 351-4; Kenzior AL, Folk WR. FEBS Lett. 1998Dec 4; 440(3): 425-9. Zn binding by Newton AL, Sharpe BK, Kwan A,transcription factors Mackay JP, Crossley M. J Biol Chem. 2000 May 19;275(20): 15128-34; Lopez Ribera I, Ruiz-Avila L, Puigdomenech P. BiochemBiophys Res Commun. 1997 Jul 18; 236(2): 510-6; de Pater S, Greco V,Pham K, Memelink J, Kijne J. Nucleic Acids Res. 1996 Dec 1; 24(23):4624-31. Synthesis of proteins to Schafer HJ, Greiner S, chelate Zn andother Rausch T, Haag-Kerwer A. metals FEBS Lett. 1997 Mar 10; 404(2-3):216-20. Rauser WE. Cell Biochem Biophys. 1999; 31(1): 19-48. Synthesisof metabolites Rauser WE. Cell Biochem Biophys. to chelate Zn and other1999; 31(1): 19-48. metals

Other biological activities that can be modulated by Zn transport,tolerance and nutrition-related genes and their products are listed inthe Reference tables. Assays for detecting such biological activitiesare described in the Protein Domain table.

Zn transport, tolerance and nutrition-related genes are differentiallytranscribed in response to low Zn concentrations. The microarraycomparison consists of probes prepared from root RNA of A. thaliana(Columbia) seedlings hydroponically grown in complete nutrient medium(control) and Zn deficient seedlings grown in −Zn nutrient medium(experimental). The data from this experiment reveal a number of typesgenes and gene products. MA_diff table reports the changes in transcriptlevels of various zinc responsive genes in entire seedlings at 1 and 6hours after a plant was sprayed with a Hoagland's solution enriched withzinc as compared to seedlings sprayed with Hoagland's solution only.

The data from this time course can be used to identify a number of typesof zinc responsive genes and gene products, including “earlyresponding,” “high zinc responders,” “repressors of zinc deprivationpathways” and “zinc deprivation responders.” Profiles of these differentzinc responsive genes are shown in the Table below with examples ofassociated biological activities.

EXAMPLES OF TRANSCRIPT PHYSIOLOGICAL BIOCHEMICAL LEVELS TYPE OF GENECONSequence ACTIVITY Upregulated Early responders to Zinc PerceptionTranscription transcripts (level at Zinc Zinc Uptake Factors 1 hour ≅ 6hours) Modulation of Zinc Transporters (level at 1 hour > 6 ResponseTransduction hours) Pathways Specific Gene Transcription Initiation ZincDeprivation Repression of Pathways Inhibit Transport of Responders toOptimize Zinc Zinc Response Pathways Degradation Level at 1 hour < 6Delayed Zinc Zinc Metabolic hours Responders Pathways Repressor of ZincNegative Regulation of Deprivation Pathways Zinc Pathways Down RegulatedEarly responder Negative Regulators of Suppressing Zinc transcripts(Level repressors of Zinc Zinc Utilization Pathways Requiring processesat 1 hour ≅ 6 utilization Pathways hours) (Level at 6 hours > 1 hour)Level at 1 hour > 6 Genes with Changes in pathways and hoursdiscontinued processes operating I cells expression or unsTable mRNAfollowing Zinc uptake

Use of Promoters of Zinc Responsive Genes

Promoters of Zinc responsive genes are useful for transcription of anydesired polynucleotide or plant or non-plant origin. Further, anydesired sequence can be transcribed in a similar temporal, tissue, orenvironmentally specific patterns as the Zinc responsive genes where thedesired sequence is operably linked to a promoter of a Zinc responsivegene. The protein product of such a polynucleotide is usuallysynthesized in the same cells, in response to the same stimuli as theprotein product of the gene from which the promoter was derived. Suchpromoter are also useful to produce antisense mRNAs to down-regulate theproduct of proteins, or to produce sense mRNAs to down-regulate mRNAsvia sense suppression.

IV. Utilities of Particular Interest

Genes capable of modulating the phenotypes in the following table areuseful produce the associated utilities in the table. Such genes can beidentified by their cDNA ID number in the Knock-in and Knock-out Tables.That is, those genes noted in those Tables to have a phenotype as listedin the following column entitled “Phenotype Modulated by a Gene” areuseful for the purpose identified in the corresponding position in thecolumn entitled “Utilities”.

Phenotype Modulated by a Gene Utilities Leaf shape Cordate decrease windopacity, Cup-shaped decrease lodging (plant fall over), Curled increasebiomass by making larger or different shaped leaves, Laceolate improvethe efficiency of mechanical harvesting, Lobed decrease transpirationfor better drought tolerance, Oval changing leaf shape to collect andabsorb water, Ovate modulation of canopy structure and shading foraltered irradiance close to the ground, Serrate enhanced uptake ofpesticides (herbicides, fungicides, etc), Trident creation of ornamentalleaf shapes, Undulate increase resistance to pathogens by decreasingamount of water that collects on leaves, Vertically Oblong changeproporation of cell types in the leaves for enhanced photosynthesis,decreased transpiration, and enhanced Other Shapes accumulation ofdesirable compounds including secondary metabolites in specializedcells, decrease insect feeding, Long petioles decrease wind opacity,Short petioles decrease lodging (plant fall over), increase biomass bybetter positioning of the leaf blade, decrease insect feeding, decreasetranspiration for better drought tolerance, position leaves mosteffectively for photosynthetic efficiency Fused ornamental applicationsto make distinctive plants, Reduced fertility Short siliques increase ordecrease the number of seeds in a fruit, increasing fruit size,modulating fruit shape to better fit harvesting or packagingrequirements, useful for controlling dehisence and seed scatter Reducedfertility useful in hybrid breeding programs, Sterility increasing fruitsize, production of seedless fruit, useful as targets for gametocides,modulating fruit shape to better fit harvesting or packagingrequirements, useful for controlling dehisence and seed scatter Flowersize useful for edible flowers useful for flower derived products suchas fragrances useful for modulating seed size and number in combinationwith seed-specific genes value in the ornamental industry Stature Largeincreasing or decreasing plant biomass, Small optimizing plant statureto increase yield under various diverse environmental conditions, e.g.,when water or nutrients are limiting, Dwarfs decreasing lodging,increasing fruit number and size, controlling shading and canopy effectsMeristems Change plant architecture, increase or decrease number ofleaves as well as change the types of leaves to increase biomass,improve photosynthetic efficiency, create new varieties of ornamentalplants with enhanced leaf design, preventing flowering to opimizevegetative growth, control of apical dominace, increase or decreaseflowering time to fit season, water or fertilizer schedules, changearrangement of leaves on the stem (phyllotaxy) to optimize plantdensity, decrease insect feeding, or decrease pathogen infection,increase number of trichome/glandular trichome producing leaves targetsfor herbicides, generate ectopic meristems and ectopic growth ofvegetative and floral tissues and seeds and fruits Stem Strong modifylignin content/composition for creation of harder woods or reducedifficulty/costs in pulping for Weak paper production or increasedigestibility of forage crops, decrease lodging, modify cell wallpolysaccharides in stems and fruits for improved texture and nutrition.increase biomass Late/Early Bolting Break the need for longvernalization of vernalization-dependent crops, e.g., winter wheat,thereby increasing yield decrease or increase generaton time increasebiomass Lethals Embryo-lethal produce seedless fruit, use as herbicidetargets Embryo-defective produce seedless fruit, use as herbicidetargets Seedling use as herbicide targets, useful for metabolicengineering, Pigment-lethals use as herbicide targets, increasephotosynthetic efficiency Pigment Dark Green Increase nutritional value,enhanced photosynthesis and carbon dioxide combustion and thereforeincrease plant vigor and biomass, enhanced photosynthetic efficiency andtherefore increase plant vigor and biomass, prolong vegetativedevelopment, enhanced protection against pathogens, YGV1 Useful astargets for herbicides, increase photosynthetic efficiency and thereforeincrease plant vigor and biomass, YGV2 Useful as targets for herbicides,control of change from embryonic to adult organs, increase metabolicefficiency, increase photosynthetic efficiency and therefore increasedplant vigor and biomass, YGV3 Useful as targets for herbicides, nitrogensensing/uptake/usage, increase metabolic efficiency and thereforeincreased plant vigor and biomass, Interveinal chlorosis to increasephotosynthetic efficiency and therefore increase plant vigor and biomassto increase or decrease nitrogen transport and therefore increase plantvigor and biomass use as herbicide targets increase metabolicefficiency, Roots Short (primary root) to access water from rainfall, toaccess rhizobia spray application, for anaerobic soils, useful tofacilitate harvest of root crops, Thick (primary root) useful forincreasing biomass of root crops, for preventing plants dislodgingduring picking and harvesting, as root grafts, for animal feedsBranching (primary root) modulation allows betters access to water,minerals, fertilizers, rhizobia prevent soil erosion, s increasing rootbiomass decrease root lodging, Long (lateral roots) modulation allowsimproved access to water, nutrients, fertilizer, rhizobia, prevent soilerosion increase root biomass decrease root lodging modulation allowscontrol on the depth of root growth in soil to access water andnutriennts modulation allows hormonal control of root growth anddevelopment (size) Agravitropic modulation allows control on the depthof root growth in soil Curling (primary root) modulation allows hormonalcontrol of root growth and development (size) useful in anaerobic soilsin allowing roots to stay close to surface harvesting of root crops Poorgermination Trichome Reduced Number Genes useful for decreasingtranspiration, Glabrous increased production of glandular trichomes foroil or other secreted chemicals of value, Increased Number use asdeterrent for insect herbivory and ovipostion modulation will increaseresistance to UV light, Wax mutants decrease insect herbivory andoviposition, compostion changes for the cosmetics industry, decreasetranspiration, provide pathogen resistance, UV protection, modulation ofleaf runoff properties and improved access for herbicides andfertilizers Cotyledons modulation of seeds structure in legumes,increase nutritional value, improve seedling competion under fieldconditions, Seeds Transparent testa genes useful for metabolicengineering anthocyanin and flavonoid pathways Light improvednutritional content Dark decrease petal abscission Flowers Otherdecrease pod shattering Hypocotysl Long to improve germination rates toimprove plant survivability Short to improve germination rates toimprove plant survivability

V. Enhanced Foods

Animals require external supplies of amino acids that they cannotsynthesize themselves. Also, some amino acids are required in largerquantities. The nutritional values of plants for animals and humans canthus be modified by regulating the amounts of the constituent aminoacids that occur as free amino acids or in proteins. For instance,higher levels of lysine and/or methionine would enhance the nutritionalvalue of corn seed. Applicants herein provide several methods formodulating the amino acid content:

-   -   (1) expressing a naturally occurring protein that has a high        percentage of the desired amino acid(s);    -   (2) expressing a modified or synthetic coding sequence that has        an enhanced percentage of the desired amino acids; or    -   (3) expressing the protein(s) that are capable of synthesizing        more of the desired amino acids.        A specific example is expressing proteins with enhanced, for        example, methionine content, preferentially in a corn or cereal        seed used for animal nutrition or in the parts of plants used        for nutritional purposes.

A protein is considered to have a high percentage of an amino acid ifthe amount of the desired amino acid is at least 1% of the total numberof residues in a protein; more preferably 2% or greater. Amino acids ofparticular interest are tryptophan, lysine, methionine, phenylalanine,threonine leucine, valine, and isoleucine. Examples of naturallyoccurring proteins with a high percentage of any one of the amino acidof particular interest are listed in the Enhanced Amino Acid Table.

The sequence(s) encoding the selected protein(s), are operably linked toa promoter and other regulatory sequences and transformed into a plantas described below. The promoter is chosen optimally for promoting thedesired level of expression of the protein in the selected organ e.g. apromoter highly functional in seeds. Modifications may be made to thesequence encoding the protein to ensure protein transport into, forexample, organelles or storage bodies or its accumulation in the organ.Such modifications may include addition of signal sequences at or nearthe N terminus and amino acid residues to modify protein stability orappropriate glycosylation. Other modifications may be made to thetranscribed nucleic acid sequence to enhance the stability ortranslatability of the mRNA, in order to ensure accumulation of more ofthe desired protein. Suitable versions of the gene construct andtransgenic plants are selected on the basis of, for example, theimproved amino acid content and nutritional value measured by standardbiochemical tests and animal feeding trials.

VI. Use of Novel Genes to Facilitate Exploitation of Plants as Factoriesfor the Synthesis of Valuable Molecules

Plants and their constituent cells, tissues, and organs are factoriesthat manufacture small organic molecules such as sugars, amino acids,fatty acids, vitamins, etc., as well as macromolecules such as proteins,nucleic acids, oils/fats and carbohydrates. Plants have long been asource of pharmaceutically beneficial chemicals; particularly, thesecondary metabolites and hormone-related molecules synthesized byplants. Plants can also be used as factories to produce carbohydrates orlipids that comprises a carbon backbone useful as precursors ofplastics, fiber, fuel, paper, pulp, rubber, solvents, lubricants,construction materials, detergents, and other cleaning materials. Plantscan also generate other compounds that are of economic value, such asdyes, flavors, and fragrances. Both the intermediates as well as theend-products of plant bio-synthetic pathways have been found useful.

With the polynucleotides and polypeptides of the instant invention,modification of both in-vitro and in-vivo synthesis of such products ispossible. One method of increasing the amount of either theintermediates or the end-products synthesized in a cell is to increasethe expression of one or more proteins in the synthesis pathway asdiscussed below. Another method of increasing production of anintermediate is to inhibit expression of protein(s) that synthesize theend-product from the intermediate. Levels of end-products andintermediates can also be modified by changing the levels of enzymesthat specifically change or degrade them. The kinds of molecules madecan be also be modified by changing the genes encoding specific enzymesperforming reactions at specific steps of the biosynthetic pathway.These genes can be from the same or a different organism. The molecularstructures in the biosynthetic pathways can thus be modified or divertedinto different branches of a pathway to make novel end-products:

Novel genes comprising selected promoters and sequences encoding enzymesare transformed into the selected plant to modify the levels,composition and/or structure of, without limitation:

-   -   Terpenoids    -   Alkaloids    -   Hormones, including brassinosteriods    -   Flavonoids    -   Steroids    -   Vitamins such as        -   Retinol        -   Riboflavin        -   Thiamine    -   Caffeine    -   Morphine and other alkaloids    -   Peptides and amino acid synthesis    -   Antioxidants    -   Starches and lipids    -   Fatty acids    -   Fructose, mannose and other sugars    -   Glycerolipid    -   Citric acid    -   Lignin    -   Flavors    -   Fragrances    -   Essential oils    -   Colors or dyes    -   Gum    -   Gels    -   Waxes

The modifications are made by designing one or more novel genes perapplication comprising promoters, to ensure production of the enzyme(s)in the relevant cells, in the right amount, and polynucleotides encodingthe relevant enzyme. The promoters and polynucleotides are the subjectof this application. The novel genes are transformed into the relevantspecies using standard procedures. Their effects are measured bystandard assays for the specific chemical/biochemical products.

These polynucleotides and proteins of the invention that participate inthe relevant pathways and are useful for changing production of theabove chemicals and biochemicals are identified in the Reference tablesby their enzyme function. More specifically, proteins of the inventionthat have the enzymatic activity of one of the entries in the followingtable entitled “Enzymes Effecting Modulation of Biological Pathways” areof interest to modulate the corresponding pathways to produce precursorsor final products noted above that are of industrial use. Biologicalactivities of particular interest are listed below.

Other polynucleotides and proteins that regulate where, when and to whatextent a pathway is active in a plant are extremely useful formodulating the synthesis and accumulation of valuable chemicals. Theseelements including transcription factors, proteins involved in signaltransduction and other proteins in the control of gene expression aredescribed elsewhere in this application.

Pathway Name Enzyme Description Comments Alkaloid Morphine 6- Also actson other alkaloids, including codeine, biosynthesis I dehydrogenasenormorphine and ethylmorphine, but only very slowly on 7,8-saturatedderivatives such as dihydromorphine and dihydrocodeine In the reversedirection, also reduces naloxone to the 6-alpha-hydroxy analog Activatedby 2- mercaptoethanol Codeinone reductase Stereospecifically catalysesthe reversible (NADPH) reduction of codeinone to codeine, which is adirect precursor of morphine in the opium poppy plant, Papaversomniferum Salutaridine reductase Stereospecifically catalyses thereversible (NADPH) reduction of salutaridine to salutaridinol, which isa direct precursor of morphinan alkaloids in the poppy plant, Papaversomniferum (S)-stylopine synthase Catalyses an oxidative reaction thatdoes not incorporate oxygen into the product Forms the secondmethylenedioxy bridge of the protoberberine alkaloid stylopine fromoxidative ring closure of adjacent phenolic and methoxy groups ofcheilanthifoline (S)-cheilanthifoline Catalyses an oxidative reactionthat does not synthase incorporate oxygen into the product Forms themethylenedioxy bridge of the protoberberine alkaloid cheilanthifolinefrom oxidative ring closure of adjacent phenolic and methoxy groups ofscoulerine Salutaridine synthase Forms the morphinan alkaloidsalutaridine by intramolecular phenol oxidation of reticuline withoutthe incorporation of oxygen into the product (S)-canadine synthaseCatalyses an oxidative reaction that does not incorporate oxygen intothe product Oxidation of the methoxyphenol group of the alkaloidtetrahydrocolumbamine results in the formation of the methylenedioxybridge of canadine Protopine 6- Involved in benzophenanthridine alkaloidmonooxygenase synthesis in higher plants Dihydrosanguinarine Involved inbenzophenanthridine alkaloid 10-monooxygenase synthesis in higher plantsMonophenol A group of copper proteins that also catalyse monooxygenasethe reaction of EC 1.10.3.1, if only 1,2- benzenediols are available assubstrate L-amino acid oxidase 1,2-dehydroreticuliniumStereospecifically reduces the 1,2- reductase (NADPH)dehydroreticulinium ion to (R)-reticuline, which is a direct precursorof morphinan alkaloids in the poppy plant, papaver somniferum The enzymedoes not catalyse the reverse reaction to any significant extent underphysiological conditions Dihydrobenzo- Also catalyzes:dihydrochelirubine + O(2) = phenanthridine oxidase chelirubine +H(2)O(2) Also catalyzes: dihydromacarpine + O(2) = macarpine + H(2)O(2)Found in higher plants Produces oxidized forms of thebenzophenanthridine alkaloids Reticuline oxidase The product of thereaction, (S)-scoulerine, is a precursor of protopine, protoberberineand benzophenanthridine alkaloid biosynthesis in plants Acts on(S)-reticuline and related compounds, converting the N-methyl group intothe methylene bridge ({grave over ( )}berberine bridge[PRIME]) of (S)-tetrahydroprotoberberines 3[PRIME]-hydroxy-N- Involved in isoquinolinealkaloid metabolism methyl-(S)-coclaurine in plants Has also been shownto catalyse the 4[PRIME]-O- methylation of (R,S)-laudanosoline, (S)-methyltransferase 3[PRIME]-hydroxycoclaurine and (R,S)-7-O-methylnoraudanosoline (S)-scoulerine 9-O- The product of this reactionis a precursor for methyltransferase protoberberine alkaloids in plantsColumbamine O- The product of this reaction is a protoberberinemethyltransferase alkaloid that is widely distributed in the plantkingdom Distinct in specificity from EC 2.1.1.88 10-hydroxydihydro- Partof the pathway for synthesis of sanguinarine 10-O- benzophenanthridinealkaloids in plants methyltransferase 12-hydroxydi- Part of the pathwayfor synthesis of hydrochelirubine 12-O- benzophenanthridine alkaloidmacarpine in methyltransferase plants (R,S)-norcoclaurine 6-Norcoclaurine is 6,7-dihydroxy-1-[(4- O-methyltransferasehydroxyphenyl)methyl]-1,2,3,4- tetrahydroisoquinoline The enzyme willalso catalyse the 6-O-methylation of (R,S)- norlaudanosoline to form6-O-methyl- norlaudanosoline, but this alkaloid has not been found tooccur in plants Salutaridinol 7-O- At higher pH values the product, 7-O-acetyltransferase acetylsalutaridinol, spontaneously closes the 4-> 5oxide bridge by allylic elimination to form the morphine precursorthebaine From the opium poppy plant, Papaver somniferum Aspartate Alsoacts on L-tyrosine, L-phenylalanine and aminotransferase L-tryptophan.This activity can be formed from EC 2.6.1.57 by controlled proteolysisTyrosine L-phenylalanine can act instead of L-tyrosine aminotransferaseThe mitochondrial enzyme may be identical with EC 2.6.1.1 The threeisoenzymic forms are interconverted by EC 3.4.22.4 Aromatic amino acidL-methionine can also act as donor, more transferase slowly Oxaloacetatecan act as acceptor Controlled proteolysis converts the enzyme to EC2.6.1.1 Tyrosine decarboxylase The bacterial enzyme also acts on 3-hydroxytyrosine and, more slowly, on 3- hydroxyphenylalanineAromatic-L-amino-acid Also acts on L-tryptophan, 5-hydroxy-L-decarboxylase tryptophan and dihydroxy-L-phenylalanine (DOPA) AlkaloidTropine dehydrogenase Oxidizes other tropan-3-alpha-ols, but not thebiosynthesis corresponding beta-derivatives II Tropinone reductaseHyoscyamine (6S)- dioxygenase 6-beta- hydroxyhyoscyamine epoxidase Amineoxidase (copper- A group of enzymes including those oxidizingcontaining) primary amines, diamines and histamine One form of EC1.3.1.15 from rat kidney also catalyses this reaction Putrescine N-methyltransferase Ornithine decarboxylase Oxalyl-CoA decarboxylasePhenylalanine May also act on L-tyrosine ammonia-lyase Androgen and3-beta-hydroxy- Acts on 3-beta-hydroxyandrost-5-en-17-one to estrogendelta(5)-steroid form androst-4-ene-3,17-dione and on 3-beta- metabolismdehydrogenase hydroxypregn-5-en-20-one to form progesterone11-beta-hydroxysteroid dehydrogenase Estradiol 17-alpha- dehydrogenase3-alpha-hydroxy-5- beta-androstane-17-one 3-alpha-dehydrogenase 3-alpha(17-beta)- Also acts on other 17-beta-hydroxysteroids, on hydroxysteroidthe 3-alpha-hydroxy group of pregnanes and dehydrogenase (NAD+) bileacids, and on benzene dihydrodiol Different from EC 1.1.1.50 or EC1.1.1.213 3-alpha-hydroxysteroid Acts on other 3-alpha-hydroxysteroidsand on dehydrogenase (B- 9-, 11- and 15-hydroxyprostaglandin B-specific) specific with respect to NAD(+) or NADP(+) (cf. EC 1.1.1.213)3(or 17)beta- Also acts on other 3-beta- or 17-beta- hydroxysteroidhydroxysteroids (cf EC 1.1.1.209) dehydrogenase Estradiol 17 beta- Alsoacts on (S)-20-hydroxypregn-4-en-3-one dehydrogenase and relatedcompounds, oxidizing the (S)-20- group B-specific with respect toNAD(P)(+) Testosterone 17-beta- dehydrogenase Testosterone 17-beta- Alsooxidizes 3-hydroxyhexobarbital to 3- dehydrogenase oxohexobarbital(NADP+) Steroid 11-beta- Also hydroxylates steroids at the 18-position,monooxygenase and converts 18-hydroxycorticosterone into aldosteroneEstradiol 6-beta- monooxygenase Androst-4-ene-3,17- Has a widespecificity A single enzyme from dione monooxygenase Cylindrocarponradicicola (EC 1.14.13.54) catalyses both this reaction and thatcatalysed by EC 1.14.99.4 3-oxo-5-alpha-steroid 4-dehydrogenase3-oxo-5-beta-steroid 4- dehydrogenase UDP- Family of enzymes accepting awide range of glucuronosyltransferase substrates, including phenols,alcohols, amines and fatty acids Some of the activities catalysed werepreviously listed separately as EC 2.4.1.42, EC 2.4.1.59, EC 2.4.1.61,EC 2.4.1.76, EC 2.4.1.77, EC 2.4.1.84, EC 2.4.1.107 and EC 2.4.1.108 Atemporary nomenclature for the various forms whose delineation is in astate of flux Steroid sulfotransferase Broad specificity resembling EC2.8.2.2, but also acts on estrone Alcohol Primary and secondaryalcohols, including sulfotransferase aliphatic alcohols, ascorbate,chloramphenicol, ephedrine and hydroxysteroids, but not phenolicsteroids, can act as acceptors (cf. EC 2.8.2.15) Estronesulfotransferase Arylsulfatase A group of enzymes with rather similarspecificities Steryl-sulfatase Also acts on some related steryl sulfates17-alpha- hydroxyprogesterone aldolase Steroid delta-isomeraseC21-Steroid 3-beta-hydroxy- Acts on 3-beta-hydroxyandrost-5-en-17-one tohormone delta(5)-steroid form androst-4-ene-3,17-dione and on 3-beta-metabolism dehydrogenase hydroxypregn-5-en-20-one to form progesterone11-beta-hydroxysteroid dehydrogenase 20-alpha- A-specific with respectto NAD(P)(+) hydroxysteroid dehydrogenase 3-alpha-hydroxysteroid Acts onother 3-alpha-hydroxysteroids and on dehydrogenase (B- 9-, 11- and15-hydroxyprostaglandin B- specific) specific with respect to NAD(+) orNADP(+) (cf. EC 1.1.1.213) 3-alpha(or 20-beta)- The 3-alpha-hydroxylgroup or 20-beta- hydroxysteroid hydroxyl group of pregnane andandrostane dehydrogenase steroids can act as donors Steroid 11-beta-Also hydroxylates steroids at the 18-position, monooxygenase andconverts 18-hydroxycorticosterone into aldosterone Corticosterone 18-monooxygenase Cholesterol The reaction proceeds in three stages, withmonooxygenase (side- hydroxylation at C-20 and C-22 preceding chaincleaving) scission of the side-chain at C-20 Steroid 21- monooxygenaseProgesterone 11-alpha- monooxygenase Steroid 17-alpha- monooxygenaseCholestenone 5-beta- reductase Cortisone beta- reductase Progesterone5-alpha- Testosterone and 20-alpha-hydroxy-4-pregnen- reductase 3-onecan act in place of progesterone 3-oxo-5-beta-steroid 4- dehydrogenaseSteroid delta-isomerase Flavonoids, Coniferyl-alcohol Specific forconiferyl alcohol; does not act on stilbene and dehydrogenase cinnamylalcohol, 4-coumaryl alcohol or lignin sinapyl alcohol biosynthesisCinnamyl-alcohol Acts on coniferyl alcohol, sinapyl alcohol, 4-dehydrogenase coumaryl alcohol and cinnamyl alcohol (cf. EC 1.1.1.194)Dihydrokaempferol 4- Also acts, in the reverse direction, on (+)-reductase dihydroquercetin and (+)-dihydromyricetin Each dihydroflavonolis reduced to the corresponding cis-flavon-3,4-diol NAD(+) can actinstead of NADP(+), more slowly Involved in the biosynthesis ofanthocyanidins in plants Flavonone 4-reductase Involved in thebiosynthesis of 3- deoxyanthocyanidins from flavonones such asnaringenin or eriodictyol Peroxidase Caffeate 3,4- dioxygenaseNaringenin 3- dioxygenase Trans-cinnamate 4- Also acts on NADH, moreslowly monooxygenase Trans-cinnamate 2- monooxygenase Flavonoid3[PRIME]- Acts on a number of flavonoids, including monooxygenasenaringenin and dihydrokaempferol Does not act on 4-coumarate or4-coumaroyl-CoA Monophenol A group of copper proteins that also catalysemonooxygenase the reaction of EC 1.10.3.1, if only 1,2- benzenediols areavailable as substrate Cinnamoyl-CoA Also acts on a number ofsubstituted reductase cinnamoyl esters of coenzyme A Caffeoyl-CoA O-methyltransferase Luteolin O- Also acts on luteolin-7-O-beta-D-glucosidemethyltransferase Caffeate O- 3,4-dihydroxybenzaldehyde and catechol canmethyltransferase act as acceptor, more slowly Apigenin 4[PRIME]-O-Converts apigenin into acacetin Naringenin methyltransferase(5,7,4[PRIME]-trihydroxyflavonone) can also act as acceptor, more slowlyQuercetin 3-O- Specific for quercetin. Related enzymes bringmethyltransferase about the 3-O-methylation of other flavonols, such asgalangin and kaempferol Isoflavone-7-O-beta- The 6-position of theglucose residue of glucoside formononetin can also act as acceptor Some6[PRIME][PRIME]-O- other 7-O-glucosides of isoflavones, flavonesmalonyltransferase and flavonols can also act, more slowly Pinosylvinsynthase Not identical with EC 2.3.1.74 or EC 2.3.1.95Naringenin-chalcone In the presence of NADH and a reductase, synthase6[PRIME]-deoxychalcone is produced Trihydroxystilbene Not identical withEC 2.3.1.74 or EC 2.3.1.146 synthase Quinate O- Caffeoyl-CoA and4-coumaroyl-CoA can also hydroxycinnamoyltransferase act as donors, moreslowly Involved in the biosynthesis of chlorogenic acid in sweet potatoand, with EC 2.3.1.98 in the formation of caffeoyl-CoA in tomatoConiferyl-alcohol Sinapyl alcohol can also act as acceptorglucosyltransferase 2-coumarate O-beta- Coumarinate(cis-2-hydroxycinnamate) does glucosyltransferase not act as acceptorScopoletin glucosyltransferase Flavonol-3-O-glucoside Converts flavonol3-O-glucosides to 3-O- L-rhamnosyltransferase rutinosides Also acts,more slowly, on rutin, quercetin 3-O-galactoside and flavonol O3-rhamnosides Flavone 7-O-beta- A number of flavones, flavonones andglucosyltransferase flavonols can function as acceptors Different fromEC 2.4.1.91 Flavonol 3-O- Acts on a variety of flavonols, includingglucosyltransferase quercetin and quercetin 7-O-glucoside Different fromEC 2.4.1.81 Flavone 7-O-beta-D-glucosides of a number ofapiosyltransferase flavonoids and of 4-substituted phenols can act asacceptors Coniferin beta- Also hydrolyzes syringin, 4-cinnamyl alcoholglucosidase beta-glucoside, and, more slowly, some other arylbeta-glycosides A plant cell-wall enzyme involved in the biosynthesis oflignin Beta-glucosidase Wide specificity for beta-D-glucosides. Someexamples also hydrolyse one or more of the following:beta-D-galactosides, alpha-L- arabinosides, beta-D-xylosides, andbeta-D- fucosides Chalcone isomerase 4-coumarate--CoA ligase

Pathway Name Enzyme Description Enzyme Comments Ascorbate and aldarateD-threo-aldose 1- Acts on L-fucose, D-arabinose and L- metabolismdehydrogenase xylose The animal enzyme was also shown to act onL-arabinose, and the enzyme from Pseudomonas caryophyllion L-glucoseL-threonate 3- dehydrogenase Glucuronate reductase Also reducesD-galacturonate May be identical with EC 1.1.1.2 Glucuronolactonereductase L-arabinose 1- dehydrogenase L-galactonolactone Acts on the1,4-lactones of L-galactonic, oxidase D-altronic, L-fuconic, D-arabinicand D- threonic acids Not identical with EC 1.1.3.8 (cf. EC 1.3.2.3)L-gulonolactone The product spontaneously isomerizes to oxidaseL-ascorbate L-ascorbate oxidase L-ascorbate peroxidase Ascorbate 2,3-dioxygenase 2,5-dioxovalerate dehydrogenase Aldehyde Wide specificity,including oxidation of dehydrogenase (NAD+) D-glucuronolactone toD-glucarate Galactonolactone Cf. EC 1.1.3.24 dehydrogenaseMonodehydroascorbate reductase (NADH) Glutathione dehydrogenase(ascorbate) L-arabinonolactonase Gluconolactonase Acts on a wide rangeof hexono-1,5- lactones Uronolactonase 1,4-lactonase Specific for1,4-lactones with 4-8 carbon atoms Does not hydrolyse simple aliphaticesters, acetylcholine, sugar lactones or substituted aliphatic lactones,e.g. 3-hydroxy-4-butyrolactone 2-dehydro-3- deoxyglucarate aldolaseL-arabinonate dehydratase Glucarate dehydratase 5-dehydro-4-deoxyglucarate dehydratase Galactarate dehydratase 2-dehydro-3-deoxy-L-arabinonate dehydratase Carbon fixation Malate dehydrogenase Alsooxidizes some other 2- hydroxydicarboxylic acids Malate dehydrogenaseDoes not decarboxylates added (decarboxylating) oxaloacetate Malatedehydrogenase Also decarboxylates added oxaloacetate (oxaloacetatedecarboxylating) (NADP+) Malate dehydrogenase Activated by light (NADP+)Glyceraldehyde-3- phosphate dehydrogenase (NADP+) (phosphorylating)Transketolase Wide specificity for both reactants, e.g. convertshydroxypyruvate and R—CHO into CO(2) and R—CHOH—CO—CH(2)OH Transketolasefrom Alcaligenes faecalis shows high activity with D-erythrose asacceptor Aspartate Also acts on L-tyrosine, L-phenylalanineaminotransferase and L-tryptophan. This activity can be formed from EC2.6.1.57 by controlled proteolysis Alanine 2-aminobutanoate acts slowlyinstead of aminotransferase alanine SedoheptulokinasePhosphoribulokinase Pyruvate kinase UTP, GTP, CTP, ITP and dATP can alsoact as donors Also phosphorylates hydroxylamine and fluoride in thepresence of CO(2) Phosphoglycerate kinase Pyruvate, phosphate dikinaseFructose- The animal enzyme also acts on bisphosphatase sedoheptulose1,7-bisphosphate Sedoheptulose- bisphosphatase Phosphoenolpyruvatecarboxylase Ribulose-bisphosphate Will utilize O(2) instead of CO(2),carboxylase forming 3-phospho-D-glycerate and 2- phosphoglycolatePhosphoenolpyruvate carboxykinase (ATP) Fructose-bisphosphate Also actson (3S,4R)-ketose 1-phosphates aldolase The yeast and bacterial enzymesare zinc proteins The enzymes increase electron- attraction by thecarbonyl group, some (Class I) forming a protonated imine with it,others (Class II), mainly of microbial origin, polarizing it with ametal ion, e.g zinc Phosphoketolase Ribulose-phosphate 3- Also convertsD-erythrose 4-phosphate epimerase into D-erythrulose 4-phosphate and D-threose 4-phosphate Triosephosphate isomerase Ribose 5-phosphate Alsoacts on D-ribose 5-diphosphate and epimerase D-ribose 5-triphosphatePhenylalanine (R)-4- Also acts, more slowly, on (R)-3- metabolismhydroxyphenyllactate phenyllactate, (R)-3-(indole-3-yl)lactatedehydrogenase and (R)-lactate Hydroxyphenyl- Also acts on 3-(3,4-pyruvate reductase dihydroxyphenyl)lactate Involved with EC 2.3.1.140 inthe biosynthesis of rosmarinic acid Aryl-alcohol A group of enzymes withbroad dehydrogenase specificity towards primary alcohols with anaromatic or cyclohex-1-ene ring, but with low or no activity towardsshort- chain aliphatic alcohols Peroxidase Catechol 1,2- Involved in themetabolism of nitro- dioxygenase aromatic compounds by a strain ofPseudomonas putida 2,3-dihydroxybenzoate 3,4-dioxygenase3-carboxyethylcatechol 2,3-dioxygenase Catechol 2,3- The enzyme fromAlcaligines sp. strain dioxygenase O-1 has also been shown to catalysethe reaction: 3-Sulfocatechol + O(2) + H(2)O = 2-hydroxymuconate +bisulfite. It has been referred to as 3-sulfocatechol 2,3- dioxygenase.Further work will be necessary to show whether or not this is a distinctenzyme 4- hydroxyphenylpyruvate dioxygenase Protocatechuate 3,4-dioxygenase Hydroxyquinol 1,2- The product isomerizes to 2- dioxygenasemaleylacetate (cis-hex-2-enedioate) Highly specific; catechol andpyrogallol are acted on at less than 1% of the rate at whichbenzene-1,2,4-triol is oxidized Protocatechuate 4,5- dioxygenasePhenylalanine 2- Also catalyses a reaction similar to that monooxygenaseof EC 1.4.3.2, forming 3-phenylpyruvate, NH(3) and H(2)O(2), but moreslowly Anthranilate 1,2- dioxygenase (deaminating, decarboxylating)Benzoate 1,2- A system, containing a reductase which dioxygenase is aniron-sulfur flavoprotein (FAD), and an iron-sulfur oxygenase Toluenedioxygenase A system, containing a reductase which is an iron-sulfurflavoprotein (FAD), an iron-sulfur oxygenase, and a ferredoxin Someother aromatic compounds, including ethylbenzene, 4-xylene and somehalogenated toluenes, are converted into the correspondingcis-dihydrodiols Naphthalene 1,2- A system, containing a reductase whichdioxygenase is an iron-sulfur flavoprotein (FAD), an iron-sulfuroxygenase, and ferredoxin Benzene 1,2- A system, containing a reductasewhich dioxygenase is an iron-sulfur flavoprotein, an iron- sulfuroxygenase and ferredoxin Salicylate 1- monooxygenase Trans-cinnamate 4-Also acts on NADH, more slowly monooxygenase Benzoate 4- monooxygenase4-hydroxybenzoate 3- Most enzymes from Pseudomonas are monooxygenasehighly specific for NAD(P)H (cf EC 1.14.13.33) 3-hydroxybenzoate 4- Alsoacts on a number of analogs of 3- monooxygenase hydroxybenzoatesubstituted in the 2, 4, 5 and 6 positions 3-hydroxybenzoate 6- Alsoacts on a number of analogs of 3- monooxygenase hydroxybenzoatesubstituted in the 2, 4, 5 and 6 positions NADPH can act instead ofNADH, more slowly 4-hydroxybenzoate 3- The enzyme from Corynebacteriummonooxygenase cyclohexanicum is highly specific for 4- (NAD(P)H)hydroxybenzoate, but uses NADH and NADPH at approximately equal rates(cf. EC 1.14.13.2). It is less specific for NADPH than EC 1.14.13.2Anthranilate 3- The enzyme from Aspergillus niger is an monooxygenaseiron protein; that from the yeast. (deaminating) Trichosporon cutaneumis a flavoprotein (FAD) Melilotate 3- monooxygenase Phenol 2- Alsoactive with resorcinol and O-cresol monooxygenase Mandelate 4-monooxygenase 3-hydroxybenzoate 2- monooxygenase 4-cresol dehydrogenasePhenazine methosulfate can act as (hydroxylating) acceptor A quinonemethide is probably formed as intermediate The product is oxidizedfurther to 4-hydroxybenzoate Benzaldehyde dehydrogenase (NAD+)Aminomuconate- Also acts on 2-hydroxymuconate semialdehyde semialdehydedehydrogenase Phenylacetaldehyde dehydrogenase 4-carboxy-2- Does not acton unsubstituted aliphatic or hydroxymuconate-6- aromatic aldehydes orglucose NAD(+) semialdehyde can replace NADP(+), but with lowerdehydrogenase affinity Aldehyde dehydrogenase (NAD(P)+) Benzaldehydedehydrogenase (NADP+) Coumarate reductase Cis-1,2- dihydrobenzene-1,2-diol dehydrogenase Cis-1,2-dihydro-1,2- Also acts, at half the rate, oncis- dihydroxynaphthalene anthracene dihydrodiol and cis- dehydrogenasephenanthrene dihydrodiol 2-enoate reductase Acts, in the reversedirection, on a wide range of alkyl and aryl alpha,beta- unsaturatedcarboxylate ions 2-butenoate was the best substrate tested Maleylacetatereductase Phenylalanine The enzyme from Bacillus badius anddehydrogenase Sporosarcina ureae are highly specific forL-phenylalanine, that from Bacillus sphaericus also acts on L-tyrosineL-amino acid oxidase Amine oxidase (flavin- Acts on primary amines, andusually also containing) on secondary and tertiary amines Amine oxidase(copper- A group of enzymes including those containing) oxidizingprimary amines, diamines and histamine One form of EC 1.3.1.15 from ratkidney also catalyses this reaction D-amino-acid Acts to some extent onall D-amino acids dehydrogenase except D-aspartate and D-glutamateAralkylamine Phenazine methosulfate can act as dehydrogenase acceptorActs on aromatic amines and, more slowly, on some long-chain aliphaticamines, but not on methylamine or ethylamine (cf EC 1.4.99.3) GlutamineN- phenylacetyltransferase Acetyl-CoA C- acyltransferase D-amino-acid N-acetyltransferase Phenylalanine N- Also acts, more slowly, onL-histidine acetyltransferase and L-alanine Glycine N- Not identicalwith EC 2.3.1.13 or EC benzoyltransferase 2.3.1.68 Aspartate Also actson L-tyrosine, L-phenylalanine aminotransferase and L-tryptophan. Thisactivity can be formed from EC 2.6.1.57 by controlled proteolysisD-alanine Acts on the D-isomers of leucine, aminotransferase aspartate,glutamate, aminobutyrate, norvaline and asparagine TyrosineL-phenylalanine can act instead of L- aminotransferase tyrosine Themitochondrial enzyme may be identical with EC 2.6.1.1 The threeisoenzymic forms are interconverted by EC 3.4.22.4 Aromatic amino acidL-methionine can also act as donor, more transferase slowly Oxaloacetatecan act as acceptor Controlled proteolysis converts the enzyme to EC2.6.1.1 Histidinol-phosphate aminotransferase 3-oxoadipate CoA-transferase 3-oxoadipate enol- Acts on the product of EC 4.1.1.44lactonase Carboxymethylene- butenolidase 2-pyrone-4,6- The productisomerizes to 4- dicarboxylate lactonase oxalmesaconate Hippuratehydrolase Acts on various N-benzoylamino acids Amidase Acylphosphatase2-hydroxymuconate- semialdehyde hydrolase Aromatic-L-amino-acid Alsoacts on L-tryptophan, 5-hydroxy-L- decarboxylase tryptophan anddihydroxy-L- phenylalanine (DOPA) Phenylpyruvate Also acts onindole-3-pyruvate decarboxylase 4-carboxymucono- lactone decarboxylaseO-pyrocatechuate decarboxylase Phenylalanine Also acts on tyrosine andother aromatic decarboxylase amino acids 4-hydroxybenzoate decarboxylaseProtocatechuate decarboxylase Benzoylformate decarboxylase4-oxalocrotonate Involved in the meta-cleavage pathway decarboxylase forthe degradation of phenols, cresols and catechols 4-hydroxy-4-methyl-2-Also acts on 4-hydroxy-4-methyl-2- oxoglutarate aldolase oxoadipate and4-carboxy-4-hydroxy-2- oxohexadioate 2-oxopent-4-enoate Also acts, moreslowly, on cis-2-oxohex- hydratase 4-enoate, but not on the trans-isomerPhenylalanine May also act on L-tyrosine ammonia-lyase Phenylalanineracemase (ATP-hydrolysing) Mandelate racemase Phenylpyruvate Also actson other arylpyruvates tautomerase 5-carboxymethyl-2- hydroxymuconatedelta-isomerase Muconolactone delta- isomerase Muconate Also acts, inthe reverse reaction, on 3- cycloisomerasemethyl-cis-cis-hexa-dienedioate and, very slowly, oncis-trans-hexadienedioate Not identical with EC 5.5.1.7 or EC 5.5.1.113-carboxy-cis,cis- muconate cycloisomerase Carboxy-cis,cis- muconatecyclase Chloromuconate Spontaneous elimination of HCl producescycloisomerase cis-4-carboxymethylenebut-2-en-4-olide Also acts inreverse direction on 2- chloro-cis,cis-muconate Not identical with EC5.5.1.1 or EC 5.5.1.11 Phenylacetate--CoA Phenoxyacetate can replacephenylacetate ligase Benzoate--CoA ligase Also acts on 2-, 3- and4-fluorobenzoate, but only very slowly on the correspondingchlorobenzoates 4-hydroxybenzoate-- CoA ligase Phenylacetate--CoA Alsoacts, more slowly, on acetate, ligase propanoate and butanoate, but noton hydroxy derivatives of phenylacetate and related compoundsPhenylalanine, tyrosine Quinate 5- and tryptophan biosynthesisdehydrogenase Shikimate 5- dehydrogenase Quinate dehydrogenase(pyrroloquinoline- quinone) Phenylalanine 4- monooxygenase PrephenateThis enzyme in the enteric bacteria also dehydrogenase possesseschorismate mutase activity (EC 5.4.99.5) and converts chorismate intoprephenate Prephenate dehydrogenase (NADP+) Cyclohexadienyl Also acts onprephenate and D- dehydrogenase prephenyllactate (cf. EC 1.3.1.12)2-methyl-branched- From Ascaris suum The reaction chain-enoyl-CoAproceeds only in the presence of another reductase flavoprotein (ETF =[PRIME]Electron- Transferring Flavoprotein[PRIME]) Phenylalanine Theenzyme from Bacillus badius and dehydrogenase Sporosarcina ureae arehighly specific for L-phenylalanine, that from Bacillus sphaericus alsoacts on L-tyrosine L-amino acid oxidase Anthranilate In some organisms,this enzyme is part of phosphoribosyl- a multifunctional proteintogether with transferase one or more components of the system forbiosynthesis of tryptophan (EC 4.1.1.48, EC 4.1.3.27, EC 4.2.1.20, andEC 5.3.1.24) 3-phosphoshikimate 1- carboxyvinyl- transferase AspartateAlso acts on L-tyrosine, L-phenylalanine aminotransferase andL-tryptophan. This activity can be formed from EC 2.6.1.57 by controlledproteolysis Tyrosine L-phenylalanine can act instead of L-aminotransferase tyrosine The mitochondrial enzyme may be identical withEC 2.6.1.1 The three isoenzymic forms are interconverted by EC 3.4.22.4Aromatic amino acid L-methionine can also act as donor, more transferaseslowly Oxaloacetate can act as acceptor Controlled proteolysis convertsthe enzyme to EC 2.6.1.1 Histidinol-phosphate aminotransferase Shikimatekinase Indole-3-glycerol- In some organisms, this enzyme is part ofphosphate synthase a multifunctional protein together with one or morecomponents of the system for biosynthesis of tryptophan (EC 2.4.2.18, EC4.1.3.27, EC 4.2.1.20, and EC 5.3.1.24) 2-dehydro-3-deoxyphosphoheptonate aldolase Anthranilate synthase In some organisms,this enzyme is part of a multifunctional protein together with one ormore components of the system for biosynthesis of tryptophan (EC2.4.2.18, EC 4.1.1.48, EC 4.2.1.20, and EC 5.3.1.24) The native enzymein the complex with uses either glutamine or (less efficiently) NH(3).The enzyme separated from the complex uses NH(3) only 3-dehydroquinatedehydratase Phosphopyruvate Also acts on 3-phospho-D-erythronatehydratase Tryptophan synthase Also catalyses the conversion of serineand indole into tryptophan and water and of indoleglycerol phosphateinto indole and glyceraldehyde phosphate In some organisms, this enzymeis part of a multifunctional protein together with one or morecomponents of the system for biosynthesis of tryptophan (EC 2.4.2.18, EC4.1.1.48, EC 4.1.3.27, and EC 5.3.1.24) Prephenate dehydratase Thisenzyme in the enteric bacteria also possesses chorismate mutase activityand converts chorismate into prephenate Carboxycyclohexadienyl Also actson prephenate and D- dehydratase prephenyllactate Cf. EC 4.2.1.513-dehydroquinate The hydrogen atoms on C-7 of the synthase substrate areretained on C-2 of the products Chorismate synthase Shikimate isnumbered so that the double-bond is between C-1 and C-2, but someearlier papers numbered in the reverse directionPhosphoribosylanthranilate In some organisms, this enzyme is part ofisomerase a multifunctional protein together with one or more componentsof the system for biosynthesis of tryptophan (EC 2.4.2.18, EC 4.1.1.48,EC 4.1.3.27, and EC 4.2.1.20) Chorismate mutase Tyrosine--tRNA ligasePhenylalanine--tRNA ligase Starch and sucrose UDP-glucose 6- Also actson UDP-2-deoxyglucose metabolism dehydrogenase Glucoside 3- The enzymeacts on D-glucose, D- dehydrogenase galactose, D-glucosides and D-galactosides, but D-glucosides react more rapidly than D-galactosidesCDP-4-dehydro-6- Two proteins are involved but no partial deoxyglucosereductase reaction has been observed in the presence of either alonePhosphorylase The recommended name should be qualified in each instanceby adding the name of the natural substance, e.g. maltodextrinphosphorylase, starch phosphorylase, glycogen phosphorylase LevansucraseSome other sugars can act as D-fructosyl acceptors Glycogen (starch) Therecommended name varies according synthase to the source of the enzymeand the nature of its synthetic product Glycogen synthase from animaltissues is a complex of a catalytic subunit and the protein glycogeninThe enzyme requires glucosylated glycogenin as a primer; this is thereaction product of EC 2.4.1.186 A similar enzyme utilizes ADP-glucose(Cf. EC 2.4.1.21) Cellulose synthase Involved in the synthesis ofcellulose A (UDP-forming) similar enzyme utilizes GDP-glucose (Cf. EC2.4.1.29) Sucrose synthase Sucrose-phosphate synthaseAlpha,alpha-trehalose- See also EC 2.4.1.36 phosphate synthase(UDP-forming) UDP- Family of enzymes accepting a wideglucuronosyltransferase range of substrates, including phenols,alcohols, amines and fatty acids Some of the activities catalysed werepreviously listed separately as EC 2.4.1.42, EC 2.4.1.59, EC 2.4.1.61,EC 2.4.1.76, EC 2.4.1.77, EC 2.4.1.84, EC 2.4.1.107 and EC 2.4.1.108 Atemporary nomenclature for the various forms whose delineation is in astate of flux 1,4-alpha-glucan Converts amylose into amylopectin Thebranching enzyme recommended name requires a qualification depending onthe product, glycogen or amylopectin, e.g. glycogen branching enzyme,amylopectin branching enzyme. The latter has frequently been termedQ-enzyme Cellobiose phosphorylase Starch (bacterial The recommended namevarious glycogen) synthase according to the source of the enzyme and thenature of its synthetic product, e.g. starch synthase, bacterialglycogen synthase A similar enzyme utilizes UDP- glucose (Cf. EC2.4.1.11) 4-alpha- An enzymic activity of this nature formsglucanotransferase part of the mammalian and Yeast glycogen branchingsystem (see EC 3.2.1.33) Cellulose synthase Involved in the synthesis ofcellulose A (GDP-forming) similar enzyme utilizes UDP-glucose (Cf. EC2.4.1.12) 1,3-beta-glucan synthase Phenol beta- Acts on a wide range ofphenols glucosyltransferase Amylosucrase Polygalacturonate 4- alpha-galacturonosyltransferase Dextransucrase Alpha,alpha-trehalosephosphorylase Sucrose phosphorylase In the forward reaction, arsenatemay replace phosphate In the reverse reaction various ketoses andL-arabinose may replace D-fructose Maltose phosphorylase1,4-beta-D-xylan synthase Hexokinase D-glucose, D-mannose, D-fructose,sorbitol and D-glucosamine can act as acceptors ITP and dATP can act asdonors The liver isoenzyme has sometimes been called glucokinasePhosphoglucokinase Glucose-1,6- D-glucose 6-phosphate can act asbisphosphate synthase acceptor, forming D-glucose 1,6- bisphosphateGlucokinase A group of enzymes found in invertebrates and microorganismshighly specific for glucose Fructokinase Glucose-1-phosphatephosphodismutase Protein-N(PI)- Comprises a group of related enzymesphosphohistidine-sugar The protein substrate is a phosphocarrierphosphotransferase protein of low molecular mass (9.5 Kd) Aphosphoenzyme intermediate is formed The enzyme translocates the sugarit phosphorylates into bacteria Aldohexoses and their glycosides andalditols are phosphorylated on O-6; fructose and sorbose on O-1 Glyceroland disaccharides are also substrates Glucose-1-phosphateadenylyltransferase Glucose-1-phosphate cytidylyltransferaseGlucose-1-phosphate Also acts, more slowly, on D-mannose 1-guanylyltransferase phosphate UTP--glucose-1- phosphateuridylyltransferase Pectinesterase Trehalose-phosphataseSucrose-phosphatase Glucose-6-phosphatase Wide distribution in animaltissues Also catalyses potent transphosphorylations from carbamoylphosphate, hexose phosphates, pyrophosphate, phosphoenolpyruvate andnucleoside di- and triphosphates, to D-glucose, D- mannose,3-methyl-D-glucose, or 2- deoxy-D-glucose (cf. EC 2.7.1.62, EC 2.7.1.79,and EC 3.9.1.1) Alpha-amylase Acts on starch, glycogen and relatedpolysaccharides and oligosaccharides in a random manner; reducing groupsare liberated in the alpha-configuration Oligo-1,6-glucosidase Alsohydrolyses palatinose The enzyme from intestinal mucosa is a singlepolypeptide chain also catalysing the reaction of EC 3.2.1.48Maltose-6[PRIME]- Hydrolyses a variety of 6-phospho-D- phosphateglucosidase glucosides, including maltose 6- phosphate,alpha[PRIME]alpha-trehalose 6-phosphate, sucrose 6-phosphate and p-nitrophenyl-alpha-D-glucopyranoside 6- phosphate (as a chromogenicsubstrate) The enzyme is activated by Fe(II), Mn(II), Co(II) and Ni(II).It is rapidly inactivated in air Polygalacturonase Beta-amylase Acts onstarch, glycogen and related polysaccharides and oligosaccharidesproducing beta-maltose by an inversion Alpha-glucosidase Group ofenzymes whose specificity is directed mainly towards the exohydrolysisof 1,4-alpha-glucosidic linkages, and that hydrolyse oligosaccharidesrapidly, relative to polysaccharides, which are hydrolysed relativelyslowly, or not at all The intestinal enzyme also hydrolysespolysaccharides, catalysing the reactions of EC 3.2.1.3, and, moreslowly, hydrolyses 1,6-alpha-D-glucose links Beta-glucosidase Widespecificity for beta-D-glucosides. Some examples also hydrolyse one ormore of the following: beta-D- galactosides, alpha-L-arabinosides, beta-D-xylosides, and beta-D-fucosides Beta-fructofuranosidase Substratesinclude sucrose Also catalyses fructotransferase reactionsAlpha,alpha-trehalase Glucan 1,4-alpha- Most forms of the enzyme canrapidly glucosidase hydrolyse 1,6-alpha-D-glucosidic bonds when the nextbond in sequence is 1,4, and some preparations of this enzyme hydrolyse1,6- and 1,3-alpha-D- glucosidic bonds in other polysaccharides Thisentry covers all such enzymes acting on polysaccharides more rapidlythan on oligosaccharides EC 3.2.1.20 from mammalian intestine cancatalyse similar reactions Beta-glucuronidase Amylo-1,6-glucosidase Inmammals and yeast this enzyme is linked to a glycosyltransferase similarto EC 2.4.1.25; together these two activities constitute the glycogendebranching system Xylan 1,4-beta- Also hydrolyses xylobiose Some otherxylosidase exoglycosidase activities have been found associated withthis enzyme in sheep liver Glucan endo-1,3-beta- Very limited action onmixed-link (1,3- D-glucosidase 1,4-)-beta-D-glucans Hydrolyseslaminarin, paramylon and pachyman Different from EC 3.2.1.6 CellulaseWill also hydrolyse 1,4-linkages in beta- D-glucans also containing1,3-linkages Sucrose alpha- This enzyme is isolated from intestinalglucosidase mucosa as a single polypeptide chain also displayingactivity towards isomaltose (oligo-1,6-glucosidase, cf. EC 3.2.1.10)Cyclomaltodextrinase Also hydrolyses linear maltodextrin Glucan1,3-beta- Acts on oligosaccharides but very slowly glucosidase onlaminaribiose Levanase Galacturan 1,4-alpha- galacturonidase Glucan1,4-beta- Acts on 1,4-beta-D-glucans and related glucosidaseoligosaccharides Cellobiose is hydrolysed, very slowly Cellulose1,4-beta- cellobiosidase Alpha,alpha- phosphotrehalase ADP-sugar Has adistinct specificity from the UDP- diphosphatase sugar pyrophosphatase(EC 3.6.1.45) Nucleotide Substrates include NAD(+), NADP(+),pyrophosphatase FAD, CoA and also ATP and ADP UDP-glucuronatedecarboxylase CDP-glucose 4,6- dehydratase CDP-abequose epimeraseUDP-glucuronate 4- epimerase Glucose-6-phosphate Also catalyses theanomerization of D- isomerase glucose 6-phosphate PhosphoglucomutaseMaximum activity is only obtained in the presence of alpha-D-glucose1,6- bisphosphate. This bisphosphate is an intermediate in the reaction,being formed by transfer of a phosphate residue from the enzyme to thesubstrate, but the dissociation of bisphosphate from the enzyme complexis much slower than the overall isomerization Also, more slowly,catalyses the interconversion of 1- phosphate and 6-phosphate isomers ofmany other alpha-D-hexoses, and the interconversion of alpha-D-ribose 1-phosphate and 5-phosphate Beta- phosphoglucomutase Maltose alpha-D-glucosyltransferase Tryptophan metabolism Indole-3-lactate dehydrogenaseIndole-3-acetaldehyde reductase (NADH) Indole-3-acetaldehyde reductase(NADPH) 3-hydroxyacyl-CoA Also oxidizes S-3-hydroxyacyl-N- dehydrogenaseacylthioethanolamine and S-3- hydroxyacylhydrolipoate Some enzymes act,more slowly, with NADP(+) Broad specificity to acyl chain-length (cf. EC1.1.1.211) O-aminophenol oxidase Isophenoxazine may be formed by asecondary condensation from the initial oxidation product Catalase Thisenzyme can also act as a peroxidase (EC 1.11.1.7) for which severalorganic substances, especially ethanol, can act as a hydrogen donor Amanganese protein containing Mn(III) in the resting state, which alsobelongs here, is often called pseudocatalase Enzymes from somemicroorganisms, such as Penicillium simplicissimum, which exhibit bothcatalase and peroxidase activity, have sometimes been referred to ascatalase- peroxidase 7,8- dihydroxykynurenate 8,8A-dioxygenaseTryptophan 2,3- Broad specificity towards tryptamine and dioxygenasederivatives including D- and L- tryptophan, 5-hydroxytryptophan andserotonin Indole 2,3-dioxygenase The enzyme from jasminum is aflavoprotein containing copper, and forms anthranilate as the finalproduct One enzyme from Tecoma stans is also a flavoprotein containingcopper and uses three atoms of oxygen per molecule of indole, to formanthranil (3,4- benzisoxazole) A second enzyme from Tecoma stans, whichis not a flavoprotein, uses four atoms of oxygen and forms anthranilateas the final product 2,3-dihydroxyindole 2,3-dioxygenaseIndoleamine-pyrrole Acts on many substituted and 2,3-dioxygenaseunsubstituted indoleamines, including melatonin Involved in thedegradation of melatonin 3-hydroxyanthranilate The product of thereaction 3,4-dioxygenase spontaneously rearrange to quinolinic acid(quin) Tryptophan 2- monooxygenase Tryptophan 2[PRIME]- Acts on a numberof indolyl-3-alkane dioxygenase derivatives, oxidizing the 3-side-chainin the 2[PRIME]-position. Best substrates are L-tryptophan and5-hydroxy-L- tryptophan Kynurenine 3- monooxygenase Unspecific Acts on awide range of substrates monooxygenase including many xenobiotics,steroids, fatty acids, vitamins and prostaglandins Reactions catalysedinclude hydroxylation, epoxidation, N-oxidation, sulfooxidation, N-, S-and O- dealkylations, desulfation, deamination, and reduction of azo,nitro, and N-oxide groups Anthranilate 3- monooxygenase Tryptophan 5-Activated by phosphorylation, catalysed monooxygenase by aCA(2+)-activated protein kinase Kynurenine 7,8- hydroxylase AldehydeWide specificity, including oxidation of dehydrogenase (NAD+)D-glucuronolactone to D-glucarate Aminomuconate- Also acts on2-hydroxymuconate semialdehyde semialdehyde dehydrogenase Aldehydeoxidase Also oxidizes quinoline and pyridine derivatives May beidentical with EC 1.1.3.22 Indole-3-acetaldehyde Also oxidizesindole-3-aldehyde and oxidase acetaldehyde, more slowly OxoglutarateComponent of the multienzyme 2- dehydrogenase oxoglutarate dehydrogenasecomplex (lipoamide) Kynurenate-7,8- dihydrodiol dehydrogenaseGlutaryl-CoA dehydrogenase L-amino acid oxidase Amine oxidase (flavin-Acts on primary amines, and usually also containing) on secondary andtertiary amines Amine oxidase (copper- A group of enzymes includingthose containing) oxidizing primary amines, diamines and histamine Oneform of EC 1.3.1.15 from rat kidney also catalyses this reactionAcetylindoxyl oxidase Acetylserotonin O- Some other hydroxyindoles alsoact as methyltransferase acceptor, more slowly Indole-3-pyruvate C-methyltransferase Amine N- A wide range of primary, secondary, andmethyltransferase tertiary amines can act as acceptors, includingtryptamine, aniline, nicotine and a variety of drugs and otherxenobiotics Aralkylamine N- Narrow specificity towards acetyltransferasearalkylamines, including serotonin Not identical with EC 2.3.1.5Acetyl-CoA C- acetyltransferase Tryptophan Also acts on5-hydroxytryptophan and, to aminotransferase a lesser extent on thephenyl amino acids Kynurenine-- Also acts on 3-hydroxykynurenineoxoglutarate aminotransferase Thioglucosidase Has a wide specificity forthioglycosides Amidase Formamidase Also acts, more slowly, on acetamide,propanamide and butanamide Arylformamidase Also acts on other aromaticformylamines Nitrilase Acts on a wide range of aromatic nitrilesincluding (indole-3-yl)-acetonitrile and also on some aliphaticnitriles, and on the corresponding acid amides (cf. EC 4.2.1.84)Kynureninase Also acts on 3[PRIME]- hydroxykynurenine and some other (3-arylcarbonyl)-alanines Aromatic-L-amino-acid Also acts on L-tryptophan,5-hydroxy-L- decarboxylase tryptophan and dihydroxy-L- phenylalanine(DOPA) Phenylpyruvate Also acts on indole-3-pyruvate decarboxylaseAminocarboxymuconate- The product rearranges non-enzymicallysemialdehyde to picolinate decarboxylase Tryptophanase Also catalysesthe synthesis of tryptophan from indole and serine Also catalyses2,3-elimination and beta- replacement reactions of some indole-substituted tryptophan analogs of L- cysteine, L-serine and other3-substituted amino acids Enoyl-CoA hydratase Acts in the reversedirection With cis- compounds, yields (3R)-3-hydroxyacyl- CoA (cf. EC4.2.1.74) Nitrile hydratase Acts on short-chain aliphatic nitriles,converting them into the corresponding acid amides Does not act on theseamides or on aromatic nitriles (cf EC 3.5.5.1) Tryptophan--tRNA ligaseTyrosine metabolism Alcohol dehydrogenase Acts on primary or secondaryalcohols or hemiacetals The animal, but not the yeast, enzyme acts alsoon cyclic secondary alcohols (R)-4- Also acts, more slowly, on (R)-3-hydroxyphenyllactate phenyllactate, (R)-3-(indole-3-yl)lactatedehydrogenase and (R)-lactate Hydroxyphenylpyruvate Also acts on 3-(3,4-reductase dihydroxyphenyl)lactate Involved with EC 2.3.1.140 in thebiosynthesis of rosmarinic acid Aryl-alcohol A group of enzymes withbroad dehydrogenase specificity towards primary alcohols with anaromatic or cyclohex-1-ene ring, but with low or no activity towardsshort- chain aliphatic alcohols Catechol oxidase Also acts on a varietyof substituted catechols Many of these enzymes also catalyse thereaction listed under EC 1.14.18.1; this is especially true for theclassical tyrosinase Iodide peroxidase 3,4- dihydroxyphenylacetate2,3-dioxygenase 4- hydroxyphenylpyruvate dioxygenase Stizolobatesynthase The intermediate product undergoes ring closure and oxidation,with NAD(P)(+) as acceptor, to stizolobic acid Stizolobinate synthaseThe intermediate product undergoes ring closure and oxidation, withNAD(P)(+) as acceptor, to stizolobinic acid Gentisate 1,2- dioxygenaseHomogentisate 1,2- dioxygenase 4-hydroxyphenylacetate Also acts on4-hydroxyhydratropate 1-monooxygenase forming 2-methylhomogentisate andon 4-hydroxyphenoxyacetate forming hydroquinone and glycolate4-hydroxyphenylacetate 3-monooxygenase Tyrosine N- monooxygenaseHydroxyphenylacetonitrile 2-monooxygenase Tyrosine 3- Activated byphosphorylation, catalysed monooxygenase by EC 2.7.1.128 Dopamine-beta-Stimulated by fumarate monooxygenase Monophenol A group of copperproteins that also monooxygenase catalyse the reaction of EC 1.10.3.1,if only 1,2-benzenediols are available as substrate Succinate-semialdehyde dehydrogenase (NAD(P)+) Aryl-aldehyde Oxidizes a number ofaromatic dehydrogenase aldehydes, but not aliphatic aldehydes AldehydeWide specificity, including oxidation of dehydrogenase (NAD+)D-glucuronolactone to D-glucarate 4-carboxy-2- Does not act onunsubstituted aliphatic or hydroxymuconate-6- aromatic aldehydes orglucose NAD(+) semialdehyde can replace NADP(+), but with lowerdehydrogenase affinity Aldehyde dehydrogenase (NAD(P)+) 4- With EC4.2.1.87, brings about the hydroxyphenylacetalde metabolism ofoctopamine in hyde dehydrogenase Pseudomonas Aldehyde oxidase Alsooxidizes quinoline and pyridine derivatives May be identical with EC1.1.3.22 L-amino acid oxidase Amine oxidase (flavin- Acts on primaryamines, and usually also containing) on secondary and tertiary aminesAmine oxidase (copper- A group of enzymes including those containing)oxidizing primary amines, diamines and histamine One form of EC 1.3.1.15from rat kidney also catalyses this reaction Aralkylamine Phenazinemethosulfate can act as dehydrogenase acceptor Acts on aromatic aminesand, more slowly, on some long-chain aliphatic amines, but not onmethylamine or ethylamine (cf EC 1.4.99.3) Phenol O- Acts on a widevariety of simple alkyl-, methyltransferase methoxy- and halo-phenolsTyramine N- Has some activity on phenylethylamine methyltransferaseanalogs Phenylethanolamine N- Acts on various phenylethanolamines;methyltransferase converts noradrenalin into adrenalin Catechol O- Themammalian enzymes act more methyltransferase rapidly on catecholaminessuch as adrenaline or noradrenaline than on catechols Glutamine N-phenylacetyltransferase Rosmarinate synthase Involved with EC 1.1.1.237in the biosynthesis of rosmarinic acid Hydroxymandelonitrile3,4-dihydroxymandelonitrile can also act glucosyltransferase as acceptorAspartate Also acts on L-tyrosine, L-phenylalanine aminotransferase andL-tryptophan. This activity can be formed from EC 2.6.1.57 by controlledproteolysis Dihydroxyphenylalanine aminotransferase TyrosineL-phenylalanine can act instead of L- aminotransferase tyrosine Themitochondrial enzyme may be identical with EC 2.6.1.1 The threeisoenzymic forms are interconverted by EC 3.4.22.4 Aromatic amino acidL-methionine can also act as donor, more transferase slowly Oxaloacetatecan act as acceptor Controlled proteolysis converts the enzyme to EC2.6.1.1 Histidinol-phosphate aminotransferase Fumarylacetoacetase Alsoacts on other 3,5- and 2,4-dioxo acids Acylpyruvate hydrolase Acts onformylpyruvate, 2,4- dioxopentanoate, 2,4-dioxohexanoate and2,4-dioxoheptanoate Tyrosine decarboxylase The bacterial enzyme alsoacts on 3- hydroxytyrosine and, more slowly, on 3- hydroxyphenylalanineAromatic-L-amino-acid Also acts on L-tryptophan, 5-hydroxy-L-decarboxylase tryptophan and dihydroxy-L- phenylalanine (DOPA) Gentisatedecarboxylase 5-oxopent-3-ene-1,2,5- tricarboxylate decarboxylaseTyrosine phenol-lyase Also slowly catalyses pyruvate formation fromD-tyrosine, S-methyl-L-cysteine, L-cysteine, L-serine and D-serine(S)-norcoclaurine The reaction makes a 6-membered ring synthase byforming a bond between C-6 of the 3,4-dihydroxyphenyl group of thedopamine and C-1 of the aldehyde in the imine formed between thesubstrates The product is the precursor of the benzylisoquinolinealkaloids in plants Will also catalyse the reaction of 4-(2-aminoethyl)benzene-1,2-diol + (3,4- dihydroxyphenyl)acetaldehyde to form(S)-norlaudanosoline, but this alkaloid has not been found to occur inplants Dihydroxyphenylalanine ammonia-lyase Phenylalanine May also acton L-tyrosine ammonia-lyase Maleylacetoacetate Also acts onmaleylpyruvate isomerase Maleylpyruvate isomerase Phenylpyruvate Alsoacts on other arylpyruvates tautomerase 5-carboxymethyl-2-hydroxymuconate delta-isomerase Tyrosine 2,3- aminomutasePhenylacetate--CoA Also acts, more slowly, on acetate, ligase propanoateand butanoate, but not on hydroxy derivatives of phenylacetate andrelated compounds

VII. Promoters as Sentinels

Useful promoters include those that are capable of facilitatingpreferential transcription, i.e. tissue-specific or developmentallyregulated gene expression and being a component of facile systems toevaluate the metabolic/physiological state of a plant cell, tissue ororgan. Many such promoters are included in this application. Operablylinking a sequence to these promoters that can act as a reporter andinserting the construct into a plant allows detection of thepreferential in plantar transcription. For example, the quantitativestate of responses to environmental conditions can be detected by usinga plant having a construct that contains a stress-inducible promoterlinked to and controlling expression of a sequence encoding GFP. Thegreater the stress promoter is induced, the greater the levels offluorescence from GFP will be produced and this provides a measure ofthe level of stress being expressed by the plant and/or the ability ofthe plant to respond internally to the stress.

More specifically, using this system the activities of any metabolicpathway (catabolic and anabolic), stress-related pathways as on anyplant gene repeated activity can be monitored. In addition, assays canbe developed using this sentinel system to select for superior genotypeswith greater yield characteristics or to select for plants with alteredresponses to chemical, herbicide, or plant growth regulators or toidentify chemical, herbicides or plant growth regulators by theirresponse on such sentinels.

Specifically, a promoter that is regulated in plants in the desired way,is operably linked to a reporter such as GFP, RFP, etc., and theconstructs are introduced into the plant of interest. The behavior ofthe reporter is monitored using technologies typically specific for thatreporter. With GFP, RFP, etc., it could typically be by microscopy ofwhole plants, organs, tissues or cells under excitation by anappropriate wavelength of UV light.

VIII. How to Make Different Embodiments of the Invention

The invention relates to (I) polynucleotides and methods of use thereof,such as

IA. Probes, Primers and Substrates;

IB. Methods of Detection and Isolation;

-   -   B.1. Hybridization;    -   B.2. Methods of Mapping;    -   B.3. Southern Blotting;    -   B.4. Isolating cDNA from Related Organisms;    -   B.5. Isolating and/or Identifying Orthologous Genes

IC. Methods of Inhibiting Gene Expression

-   -   C.1. Antisense    -   C.2. Ribozyme Constructs;    -   C.3. Chimeraplasts;    -   C.4 Co-Suppression;    -   C.5. Transcriptional Silencing    -   C.6. Other Methods to Inhibit Gene Expression

ID. Methods of Functional Analysis;

IE. Promoter Sequences and Their Use;

IF. UTRs and/or Intron Sequences and Their Use; and

IG. Coding Sequences and Their Use.

The invention also relates to (II) polypeptides and proteins and methodsof use thereof, such as

IIA. Native Polypeptides and Proteins

-   -   A.1 Antibodies    -   A.2 In Vitro Applications

IIB. Polypeptide Variants, Fragments and Fusions

-   -   B.1 Variants    -   B.2 Fragments    -   B.3 Fusions

The invention also includes (III) methods of modulating polypeptideproduction, such as

IIIA. Suppression

-   -   A.1 Antisense    -   A.2 Ribozymes    -   A.3 Co-suppression    -   A.4 Insertion of Sequences into the Gene to be Modulated    -   A.5 Promoter Modulation    -   A.6 Expression of Genes containing Dominant-Negative Mutations

IIIB. Enhanced Expression

-   -   B.1 Insertion of an Exogenous Gene    -   B.2 Promoter Modulation

The invention further concerns (IV) gene constructs and vectorconstruction, such as

IVA. Coding Sequences

IVB. Promoters

IVC. Signal Peptides

The invention still further relates to

V. Transformation Techniques

I. Polynucleotides

Exemplified SDFs of the invention represent fragments of the genome ofcorn, wheat, rice, soybean or Arabidopsis and/or represent mRNAexpressed from that genome. The isolated nucleic acid of the inventionalso encompasses corresponding fragments of the genome and/or cDNAcomplement of other organisms as described in detail below.

Polynucleotides of the invention can be isolated from polynucleotidelibraries using primers comprising sequences similar to those described,in the attached Reference, Sequences Protein Group, and Protein GroupMatrix Tables or complements thereof. See, for example, the methodsdescribed in Sambrook et al., supra.

Alternatively, the polynucleotides of the invention can be produced bychemical synthesis. Such synthesis methods are described below.

It is contemplated that the nucleotide sequences presented herein maycontain some small percentage of errors. These errors may arise in thenormal course of determination of nucleotide sequences. Sequence errorscan be corrected by obtaining seeds deposited under the accessionnumbers cited above, propagating them, isolating genomic DNA orappropriate mRNA from the resulting plants or seeds thereof, amplifyingthe relevant fragment of the genomic DNA or mRNA using primers having asequence that flanks the erroneous sequence, and sequencing theamplification product.

I.A. Probes, Primers and Substrates

SDFs of the invention can be applied to substrates for use in arrayapplications such as, but not limited to, assays of global geneexpression, for example under varying conditions of development, growthconditions. The arrays can also be used in diagnostic or forensicmethods (WO95/35505, U.S. Pat. No. 5,445,943 and U.S. Pat. No.5,410,270).

Probes and primers of the instant invention will hybridize to apolynucleotide comprising a sequence in or encoded by those in theReference, Sequence, Protein Group, and Protein Group Matrix tables orfragments or complement thereof. Though many different nucleotidesequences can encode an amino acid sequence, the sequences of thereference and Sequence table or sequences that encode polypeptides orfragments thereof described in Protein Group and Protein Group Matrixtables are generally preferred for encoding polypeptides of theinvention. However, the sequence of the probes and/or primers of theinstant invention need not be identical to those in the Reference andSequence tables or the complements thereof. For example, some variationin probe or primer sequence and/or length can allow additional familymembers to be detected, as well as orthologous genes and moretaxonomically distant related sequences. Similarly, probes and/orprimers of the invention can include additional nucleotides that serveas a label for detecting the formed duplex or for subsequent cloningpurposes.

Probe length will vary depending on the application. For use as primers,probes are 12-40 nucleotides, preferably 18-30 nucleotides long. For usein mapping, probes are preferably 50 to 500 nucleotides, preferably100-250 nucleotides long. For Southern hybridizations, probes as long asseveral kilobases can be used as explained below.

The probes and/or primers can be produced by synthetic procedures suchas the triester method of Matteucci et al. J. Am. Chem. Soc. 103:3185(1981); or according to Urdea et al. Proc. Natl. Acad. 80:7461 (1981) orusing commercially available automated oligonucleotide synthesizers.

I.B. Methods of Detection and Isolation

The polynucleotides of the invention can be utilized in a number ofmethods known to those skilled in the art as probes and/or primers toisolate and detect polynucleotides, including, without limitation:Southerns, Northerns, Branched DNA hybridization assays, polymerasechain reaction, and microarray assays, and variations thereof. Specificmethods given by way of examples, and discussed below include:

Hybridization

Methods of Mapping

Southern Blotting

Isolating cDNA from Related Organisms

Isolating and/or Identifying Orthologous Genes.

Also, the nucleic acid molecules of the invention can used in othermethods, such as high density oligonucleotide hybridizing assays,described, for example, in U.S. Pat. Nos. 6,004,753; 5,945,306;5,945,287; 5,945,308; 5,919,686; 5,919,661; 5,919,627; 5,874,248;5,871,973; 5,871,971; and 5,871,930; and PCT Pub. Nos. WO 9946380; WO9933981; WO 9933870; WO 9931252; WO 9915658; WO 9906572; WO 9858052; WO9958672; and WO 9810858.

B.1. Hybridization

The isolated SDFs of the Reference and Sequence tables or SDFs encodingpolypeptides of the Protein Group and Protein Group Matrix tables orfragments thereof of the present invention can be used as probes and/orprimers for detection and/or isolation of related polynucleotidesequences through hybridization. Hybridization of one nucleic acid toanother constitutes a physical property that defines the subject SDF ofthe invention and the identified related sequences. Also, suchhybridization imposes structural limitations on the pair. A good generaldiscussion of the factors for determining hybridization conditions isprovided by Sambrook et al. (“Molecular Cloning, a Laboratory Manual,2nd ed., c. 1989 by Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; see esp., chapters 11 and 12). Additional considerationsand details of the physical chemistry of hybridization are provided byG. H. Keller and M. M. Manak “DNA Probes”, 2^(nd) Ed. pp. 1-25, c. 1993by Stockton Press, New York, N.Y.

Depending on the stringency of the conditions under which these probesand/or primers are used, polynucleotides exhibiting a wide range ofsimilarity to those in the Reference and Sequence or encodingpolypeptides of the Protein Group and Protein Group Matrix tables orfragments thereof can be detected or isolated. When the practitionerwishes to examine the result of membrane hybridizations under a varietyof stringencies, an efficient way to do so is to perform thehybridization under a low stringency condition, then to wash thehybridization membrane under increasingly stringent conditions.

When using SDFs to identify orthologous genes in other species, thepractitioner will preferably adjust the amount of target DNA of eachspecies so that, as nearly as is practical, the same number of genomeequivalents are present for each species examined. This prevents faintsignals from species having large genomes, and thus small numbers ofgenome equivalents per mass of DNA, from erroneously being interpretedas absence of the corresponding gene in the genome.

The probes and/or primers of the instant invention can also be used todetect or isolate nucleotides that are “identical” to the probes orprimers. Two nucleic acid sequences or polypeptides are said to be“identical” if the sequence of nucleotides or amino acid residues,respectively, in the two sequences is the same when aligned for maximumcorrespondence as described below.

Isolated polynucleotides within the scope of the invention also includeallelic variants of the specific sequences presented in the Reference,Sequence, Protein Group, and Protein Group Matrix tables. The probesand/or primers of the invention can also be used to detect and/orisolate polynucleotides exhibiting at least 80% sequence identity withthe sequences of the reference, Sequence or encoding polypeptides of theProtein Group and Protein Group Matrix tables or fragments thereof.

With respect to nucleotide sequences, degeneracy of the genetic codeprovides the possibility to substitute at least one base of the basesequence of a gene with a different base without causing the amino acidsequence of the polypeptide produced from the gene to be changed. Hence,the DNA of the present invention may also have any base sequence thathas been changed from a sequence in the Reference, Sequence, ProteinGroup, and Protein Group Matrix tables by substitution in accordancewith degeneracy of genetic code. References describing codon usageinclude: Carels et al., J. Mol. Evol. 46: 45 (1998) and Fennoy et al.,Nucl. Acids Res. 21(23): 5294 (1993).

B.2. Mapping

The isolated SDF DNA of the invention can be used to create varioustypes of genetic and physical maps of the genome of corn, Arabidopsis,soybean, rice, wheat, or other plants. Some SDFs may be absolutelyassociated with particular phenotypic traits, allowing construction ofgross genetic maps. While not all SDFs will immediately be associatedwith a phenotype, all SDFs can be used as probes for identifyingpolymorphisms associated with phenotypes of interest. Briefly, onemethod of mapping involves total DNA isolation from individuals. It issubsequently cleaved with one or more restriction enzymes, separatedaccording to mass, transferred to a solid support, hybridized with SDFDNA and the pattern of fragments compared. Polymorphisms associated witha particular SDF are visualized as differences in the size of fragmentsproduced between individual DNA samples after digestion with aparticular restriction enzyme and hybridization with the SDF. Afteridentification of polymorphic SDF sequences, linkage studies can beconducted. By using the individuals showing polymorphisms as parents incrossing programs, F2 progeny recombinants or recombinant inbreds, forexample, are then analyzed. The order of DNA polymorphisms along thechromosomes can be determined based on the frequency with which they areinherited together versus independently. The closer two polymorphismsare together in a chromosome the higher the probability that they areinherited together. Integration of the relative positions of all thepolymorphisms and associated marker SDFs can produce a genetic map ofthe species, where the distances between markers reflect therecombination frequencies in that chromosome segment.

The use of recombinant inbred lines for such genetic mapping isdescribed for Arabidopsis by Alonso-Blanco et al. (Methods in MolecularBiology, vol. 82, “Arabidopsis Protocols”, pp. 137-146, J. M.Martinez-Zapater and J. Salinas, eds., c. 1998 by Humana Press, Totowa,N.J.) and for corn by Burr (“Mapping Genes with Recombinant Inbreds”,pp. 249-254. In Freeling, M. and V. Walbot (Ed.), The Maize Handbook, c.1994 by Springer-Verlag New York, Inc.: New York, N.Y., USA; BerlinGermany; Burr et al. Genetics (1998) 118: 519; Gardiner, J. et al.,(1993) Genetics 134: 917). This procedure, however, is not limited toplants and can be used for other organisms (such as yeast) or forindividual cells.

The SDFs of the present invention can also be used for simple sequencerepeat (SSR) mapping. Rice SSR mapping is described by Morgante et al.(The Plant Journal (1993) 3: 165), Panaud et al. (Genome (1995) 38:1170); Senior et al. (Crop Science (1996) 36: 1676), Taramino et al.(Genome (1996) 39: 277) and Ahn et al. (Molecular and General Genetics(1993) 241: 483-90). SSR mapping can be achieved using various methods.In one instance, polymorphisms are identified when sequence specificprobes contained within an SDF flanking an SSR are made and used inpolymerase chain reaction (PCR) assays with template DNA from two ormore individuals of interest. Here, a change in the number of tandemrepeats between the SSR-flanking sequences produces differently sizedfragments (U.S. Pat. No. 5,766,847). Alternatively, polymorphisms can beidentified by using the PCR fragment produced from the SSR-flankingsequence specific primer reaction as a probe against Southern blotsrepresenting different individuals (U. H. Refseth et al., (1997)Electrophoresis 18: 1519).

Genetic and physical maps of crop species have many uses. For example,these maps can be used to devise positional cloning strategies forisolating novel genes from the mapped crop species. In addition, becausethe genomes of closely related species are largely syntenic (that is,they display the same ordering of genes within the genome), these mapscan be used to isolate novel alleles from relatives of crop species bypositional cloning strategies.

The various types of maps discussed above can be used with the SDFs ofthe invention to identify Quantitative Trait Loci (QTLs). Many importantcrop traits, such as the solids content of tomatoes, are quantitativetraits and result from the combined interactions of several genes. Thesegenes reside at different loci in the genome, oftentimes on differentchromosomes, and generally exhibit multiple alleles at each locus. TheSDFs of the invention can be used to identify QTLs and isolate specificalleles as described by de Vicente and Tanksley (Genetics 134:585(1993)). In addition to isolating QTL alleles in present crop species,the SDFs of the invention can also be used to isolate alleles from thecorresponding QTL of wild relatives. Transgenic plants having variouscombinations of QTL alleles can then be created and the effects of thecombinations measured. Once a desired allele combination has beenidentified, crop improvement can be accomplished either throughbiotechnological means or by directed conventional breeding programs(for review see Tanksley and McCouch, Science 277:1063 (1997)).

In another embodiment, the SDFs can be used to help create physical mapsof the genome of corn, Arabidopsis and related species. Where SDFs havebeen ordered on a genetic map, as described above, they can be used asprobes to discover which clones in large libraries of plant DNAfragments in YACs, BACs, etc. contain the same SDF or similar sequences,thereby facilitating the assignment of the large DNA fragments tochromosomal positions. Subsequently, the large BACs, YACs, etc. can beordered unambiguously by more detailed studies of their sequencecomposition (e.g. Marra et al. (1997) Genomic Research 7:1072-1084) andby using their end or other sequences to find the identical sequences inother cloned DNA fragments. The overlapping of DNA sequences in this wayallows large contigs of plant sequences to be built that, whensufficiently extended, provide a complete physical map of a chromosome.Sometimes the SDFs themselves will provide the means of joining clonedsequences into a contig.

The patent publication WO95/35505 and U.S. Pat. Nos. 5,445,943 and5,410,270 describe scanning multiple alleles of a plurality of lociusing hybridization to arrays of oligonucleotides. These techniques areuseful for each of the types of mapping discussed above.

Following the procedures described above and using a plurality of theSDFs of the present invention, any individual can be genotyped. Theseindividual genotypes can be used for the identification of particularcultivars, varieties, lines, ecotypes and genetically modified plants orcan serve as tools for subsequent genetic studies involving multiplephenotypic traits.

B.3 Southern Blot Hybridization

The sequences from Reference and Sequence and those encodingpolypeptides of Protein Group and Protein Group Matrix tables orfragments thereof can be used as probes for various hybridizationtechniques. These techniques are useful for detecting targetpolynucleotides in a sample or for determining whether transgenicplants, seeds or host cells harbor a gene or sequence of interest andthus might be expected to exhibit a particular trait or phenotype.

In addition, the SDFs from the invention can be used to isolateadditional members of gene families from the same or different speciesand/or orthologous genes from the same or different species. This isaccomplished by hybridizing an SDF to, for example, a Southern blotcontaining the appropriate genomic DNA or cDNA. Given the resultinghybridization data, one of ordinary skill in the art could distinguishand isolate the correct DNA fragments by size, restriction sites,sequence and stated hybridization conditions from a gel or from alibrary.

Identification and isolation of orthologous genes from closely relatedspecies and alleles within a species is particularly desirable becauseof their potential for crop improvement. Many important crop traits,such as the solid content of tomatoes, result from the combinedinteractions of the products of several genes residing at different lociin the genome. Generally, alleles at each of these loci can makequantitative differences to the trait. By identifying and isolatingnumerous alleles for each locus from within or different species,transgenic plants with various combinations of alleles can be createdand the effects of the combinations measured. Once a more favorableallele combination has been identified, crop improvement can beaccomplished either through biotechnological means or by directedconventional breeding programs (Tanksley et al. Science 277:1063(1997)).

The results from hybridizations of the SDFs of the invention to, forexample, Southern blots containing DNA from another species can also beused to generate restriction fragment maps for the corresponding genomicregions. These maps provide additional information about the relativepositions of restriction sites within fragments, further distinguishingmapped DNA from the remainder of the genome.

Physical maps can be made by digesting genomic DNA with differentcombinations of restriction enzymes.

Probes for Southern blotting to distinguish individual restrictionfragments can range in size from 15 to 20 nucleotides to severalthousand nucleotides. More preferably, the probe is 100 to 1,000nucleotides long for identifying members of a gene family when it isfound that repetitive sequences would complicate the hybridization. Foridentifying an entire corresponding gene in another species, the probeis more preferably the length of the gene, typically 2,000 to 10,000nucleotides, but probes 50-1,000 nucleotides long might be used. Somegenes, however, might require probes up to 1,500 nucleotides long oroverlapping probes constituting the full-length sequence to span theirlengths.

Also, while it is preferred that the probe be homogeneous with respectto its sequence, it is not necessary. For example, as described below, aprobe representing members of a gene family having diverse sequences canbe generated using PCR to amplify genomic DNA or RNA templates usingprimers derived from SDFs that include sequences that define the genefamily.

For identifying corresponding genes in another species, the next mostpreferable probe is a cDNA spanning the entire coding sequence, whichallows all of the mRNA-coding fragment of the gene to be identified.Probes for Southern blotting can easily be generated from SDFs by makingprimers having the sequence at the ends of the SDF and using corn orArabidopsis genomic DNA as a template. In instances where the SDFincludes sequence conserved among species, primers including theconserved sequence can be used for PCR with genomic DNA from a speciesof interest to obtain a probe.

Similarly, if the SDF includes a domain of interest, that fragment ofthe SDF can be used to make primers and, with appropriate template DNA,used to make a probe to identify genes containing the domain.Alternatively, the PCR products can be resolved, for example by gelelectrophoresis, and cloned and/or sequenced. Using Southernhybridization, the variants of the domain among members of a genefamily, both within and across species, can be examined.

B.4.1 Isolating DNA from Related Organisms

The SDFs of the invention can be used to isolate the corresponding DNAfrom other organisms. Either cDNA or genomic DNA can be isolated. Forisolating genomic DNA, a lambda, cosmid, BAC or YAC, or other largeinsert genomic library from the plant of interest can be constructedusing standard molecular biology techniques as described in detail bySambrook et al. 1989 (Molecular Cloning: A Laboratory Manual, 2^(nd) ed.Cold Spring Harbor Laboratory Press, New York) and by Ausubel et al.1992 (Current Protocols in Molecular Biology, Greene Publishing, NewYork).

To screen a phage library, for example, recombinant lambda clones areplated out on appropriate bacterial medium using an appropriate E. colihost strain. The resulting plaques are lifted from the plates usingnylon or nitrocellulose filters. The plaque lifts are processed throughdenaturation, neutralization, and washing treatments following thestandard protocols outlined by Ausubel et al. (1992). The plaque liftsare hybridized to either radioactively labeled or non-radioactivelylabeled SDF DNA at room temperature for about 16 hours, usually in thepresence of 50% formamide and 5×SSC (sodium chloride and sodium citrate)buffer and blocking reagents. The plaque lifts are then washed at 42° C.with 1% Sodium Dodecyl Sulfate (SDS) and at a particular concentrationof SSC. The SSC concentration used is dependent upon the stringency atwhich hybridization occurred in the initial Southern blot analysisperformed. For example, if a fragment hybridized under medium stringency(e.g., Tm−20° C.), then this condition is maintained or preferablyadjusted to a less stringent condition (e.g., Tm−30° C.) to wash theplaque lifts. Positive clones show detectable hybridization e.g., byexposure to X-ray films or chromogen formation. The positive clones arethen subsequently isolated for purification using the same generalprotocol outlined above. Once the clone is purified, restrictionanalysis can be conducted to narrow the region corresponding to the geneof interest. The restriction analysis and succeeding subcloning stepscan be done using procedures described by, for example Sambrook et al.(1989) cited above.

The procedures outlined for the lambda library are essentially similarto those used for YAC library screening, except that the YAC clones areharbored in bacterial colonies. The YAC clones are plated out atreasonable density on nitrocellulose or nylon filters supported byappropriate bacterial medium in petri plates. Following the growth ofthe bacterial clones, the filters are processed through thedenaturation, neutralization, and washing steps following the proceduresof Ausubel et al. 1992. The same hybridization procedures for lambdalibrary screening are followed.

To isolate cDNA, similar procedures using appropriately modified vectorsare employed. For instance, the library can be constructed in a lambdavector appropriate for cloning cDNA such as λgt11. Alternatively, thecDNA library can be made in a plasmid vector. cDNA for cloning can beprepared by any of the methods known in the art, but is preferablyprepared as described above. Preferably, a cDNA library will include ahigh proportion of full-length clones.

B. 5. Isolating and/or Identifying Orthologous Genes

Probes and primers of the invention can be used to identify and/orisolate polynucleotides related to those in the Reference, Sequence,Protein Group, and Protein Group Matrix tables. Related polynucleotidesare those that are native to other plant organisms and exhibit eithersimilar sequence or encode polypeptides with similar biologicalactivity. One specific example is an orthologous gene. Orthologous geneshave the same functional activity. As such, orthologous genes may bedistinguished from homologous genes. The percentage of identity is afunction of evolutionary separation and, in closely related species, thepercentage of identity can be 98 to 100%. The amino acid sequence of aprotein encoded by an orthologous gene can be less than 75% identical,but tends to be at least 75% or at least 80% identical, more preferablyat least 90%, most preferably at least 95% identical to the amino acidsequence of the reference protein.

To find orthologous genes, the probes are hybridized to nucleic acidsfrom a species of interest under low stringency conditions, preferablyone where sequences containing as much as 40-45% mismatches will be ableto hybridize. This condition is established by T_(m)−40° C. to Tm−48° C.(see below). Blots are then washed under conditions of increasingstringency. It is preferable that the wash stringency be such thatsequences that are 85 to 100% identical will hybridize. More preferably,sequences 90 to 100% identical will hybridize and most preferably onlysequences greater than 95% identical will hybridize. One of ordinaryskill in the art will recognize that, due to degeneracy in the geneticcode, amino acid sequences that are identical can be encoded by DNAsequences as little as 67% identical or less. Thus, it is preferable,for example, to make an overlapping series of shorter probes, on theorder of 24 to 45 nucleotides, and individually hybridize them to thesame arrayed library to avoid the problem of degeneracy introducinglarge numbers of mismatches.

As evolutionary divergence increases, genome sequences also tend todiverge. Thus, one of skill will recognize that searches for orthologousgenes between more divergent species will require the use of lowerstringency conditions compared to searches between closely relatedspecies. Also, degeneracy of the genetic code is more of a problem forsearches in the genome of a species more distant evolutionarily from thespecies that is the source of the SDF probe sequences.

Therefore the method described in Bouckaert et al., U.S. Ser. No.60/121,700 Atty. Dkt. No. 2750-117P, Client Dkt. No. 00010.001, filedFeb. 25, 1999, hereby incorporated in its entirety by reference, can beapplied to the SDFs of the present invention to isolate related genesfrom plant species which do not hybridize to the corn Arabidopsis,soybean, rice, wheat, and other plant sequences of the reference,Sequence, Protein Group, and Protein Group Matrix tables.

Identification of the relationship of nucleotide or amino acid sequencesamong plant species can be done by comparing the nucleotide or aminoacid sequences of SDFs of the present application with nucleotide oramino acid sequences of other SDFs such as those present in applicationslisted in the table below:

The SDFs of the invention can also be used as probes to search for genesthat are related to the SDF within a species. Such related genes aretypically considered to be members of a gene family. In such a case, thesequence similarity will often be concentrated into one or a fewfragments of the sequence. The fragments of similar sequence that definethe gene family typically encode a fragment of a protein or RNA that hasan enzymatic or structural function. The percentage of identity in theamino acid sequence of the domain that defines the gene family ispreferably at least 70%, more preferably 80 to 95%, most preferably 85to 99%. To search for members of a gene family within a species, a lowstringency hybridization is usually performed, but this will depend uponthe size, distribution and degree of sequence divergence of domains thatdefine the gene family. SDFs encompassing regulatory regions can be usedto identify coordinately expressed genes by using the regulatory regionsequence of the SDF as a probe.

In the instances where the SDFs are identified as being expressed fromgenes that confer a particular phenotype, then the SDFs can also be usedas probes to assay plants of different species for those phenotypes.

I.C. Methods to Inhibit Gene Expression

The nucleic acid molecules of the present invention can be used toinhibit gene transcription and/or translation. Example of such methodsinclude, without limitation:

Antisense Constructs;

Ribozyme Constructs;

Chimeraplast Constructs;

Co-Suppression;

Transcriptional Silencing; and

Other Methods of Gene Expression.

C.1 Antisense

In some instances it is desirable to suppress expression of anendogenous or exogenous gene. A well-known instance is the FLAVOR-SAVOR™tomato, in which the gene encoding ACC synthase is inactivated by anantisense approach, thus delaying softening of the fruit after ripening.See for example, U.S. Pat. No. 5,859,330; U.S. Pat. No. 5,723,766;Oeller, et al, Science, 254:437-439 (1991); and Hamilton et al, Nature,346:284-287 (1990). Also, timing of flowering can be controlled bysuppression of the FLOWERING LOCUS C (FLC); high levels of thistranscript are associated with late flowering, while absence of FLC isassociated with early flowering (S. D. Michaels et al., Plant Cell11:949 (1999). Also, the transition of apical meristem from productionof leaves with associated shoots to flowering is regulated by TERMINALFLOWER1, APETALA1 and LEAFY. Thus, when it is desired to induce atransition from shoot production to flowering, it is desirable tosuppress TFL1 expression (S. J. Liljegren, Plant Cell 11:1007 (1999)).As another instance, arrested ovule development and female sterilityresult from suppression of the ethylene forming enzyme but can bereversed by application of ethylene (D. De Martinis et al., Plant Cell11:1061 (1999)). The ability to manipulate female fertility of plants isuseful in increasing fruit production and creating hybrids.

In the case of polynucleotides used to inhibit expression of anendogenous gene, the introduced sequence need not be perfectly identicalto a sequence of the target endogenous gene. The introducedpolynucleotide sequence will typically be at least substantiallyidentical to the target endogenous sequence.

Some polynucleotide SDFs in the Reference, Sequence, Protein Group, andProtein Group Matrix tables represent sequences that are expressed incorn, wheat, rice, soybean Arabidopsis and/or other plants. Thus theinvention includes using these sequences to generate antisenseconstructs to inhibit translation and/or degradation of transcripts ofsaid SDFs, typically in a plant cell.

To accomplish this, a polynucleotide segment from the desired gene thatcan hybridize to the mRNA expressed from the desired gene (the“antisense segment”) is operably linked to a promoter such that theantisense strand of RNA will be transcribed when the construct ispresent in a host cell. A regulated promoter can be used in theconstruct to control transcription of the antisense segment so thattranscription occurs only under desired circumstances.

The antisense segment to be introduced generally will be substantiallyidentical to at least a fragment of the endogenous gene or genes to berepressed. The sequence, however, need not be perfectly identical toinhibit expression. Further, the antisense product may hybridize to theuntranslated region instead of or in addition to the coding sequence ofthe gene. The vectors of the present invention can be designed such thatthe inhibitory effect applies to other proteins within a family of genesexhibiting homology or substantial homology to the target gene.

For antisense suppression, the introduced antisense segment sequencealso need not be full length relative to either the primarytranscription product or the fully processed mRNA. Generally, a higherpercentage of sequence identity can be used to compensate for the use ofa shorter sequence. Furthermore, the introduced sequence need not havethe same intron or exon pattern, and homology of non-coding segments maybe equally effective. Normally, a sequence of between about 30 or 40nucleotides and the full length of the transcript can be used, though asequence of at least about 100 nucleotides is preferred, a sequence ofat least about 200 nucleotides is more preferred, and a sequence of atleast about 500 nucleotides is especially preferred.

C.2. Ribozymes

It is also contemplated that gene constructs representing ribozymes andbased on the SDFs in the Reference and Sequence tables or those encodingpolypeptides of the Protein Group and Protein Group Matrix tables andfragment thereof are an object of the invention. Ribozymes can also beused to inhibit expression of genes by suppressing the translation ofthe mRNA into a polypeptide. It is possible to design ribozymes thatspecifically pair with virtually any target RNA and cleave thephosphodiester backbone at a specific location, thereby functionallyinactivating the target RNA. In carrying out this cleavage, the ribozymeis not itself altered, and is thus capable of recycling and cleavingother molecules, making it a true enzyme. The inclusion of ribozymesequences within antisense RNAs confers RNA-cleaving activity upon them,thereby increasing the activity of the constructs.

A number of classes of ribozymes have been identified. One class ofribozymes is derived from a number of small circular RNAs, which arecapable of self-cleavage and replication in plants. The RNAs replicateeither alone (viroid RNAs) or with a helper virus (satellite RNAs).Examples include RNAs from avocado sunblotch viroid and the satelliteRNAs from tobacco ringspot virus, lucerne transient streak virus, velvettobacco mottle virus, solanum nodiflorum mottle virus and subterraneanclover mottle virus. The design and use of target RNA-specific ribozymesis described in Haseloff et al. Nature, 334:585 (1988).

Like the antisense constructs above, the ribozyme sequence fragmentnecessary for pairing need not be identical to the target nucleotides tobe cleaved, nor identical to the sequences in the Reference and Sequencetables or those encoding polypeptide of the Protein Group and ProteinGroup Matrix tables or fragments thereof. Ribozymes may be constructedby combining the ribozyme sequence and some fragment of the target genewhich would allow recognition of the target gene mRNA by the resultingribozyme molecule. Generally, the sequence in the ribozyme capable ofbinding to the target sequence exhibits a percentage of sequenceidentity with at least 80%, preferably with at least 85%, morepreferably with at least 90% and most preferably with at least 95%, evenmore preferably, with at least 96%, 97%, 98% or 99% sequence identity tosome fragment of a sequence in the Reference, Sequence, Protein Group,and Protein Group Matrix tables or the complement thereof. The ribozymecan be equally effective in inhibiting mRNA translation by cleavingeither in the untranslated or coding regions. Generally, a higherpercentage of sequence identity can be used to compensate for the use ofa shorter sequence. Furthermore, the introduced sequence need not havethe same intron or exon pattern, and homology of non-coding segments maybe equally effective.

C.3. Chimeraplasts

The SDFs of the invention, such as those described by Reference,Sequence, Protein Group, and Protein Group Matrix tables, can also beused to construct chimeraplasts that can be introduced into a cell toproduce at least one specific nucleotide change in a sequencecorresponding to the SDF of the invention. A chimeraplast is anoligonucleotide comprising DNA and/or RNA that specifically hybridizesto a target region in a manner which creates a mismatched base-pair.This mismatched base-pair signals the cell's repair enzyme machinerywhich acts on the mismatched region resulting in the replacement,insertion or deletion of designated nucleotide(s). The altered sequenceis then expressed by the cell's normal cellular mechanisms.Chimeraplasts can be designed to repair mutant genes, modify genes,introduce site-specific mutations, and/or act to interrupt or alternormal gene function (U.S. Pat. Nos. 6,010,907 and 6,004,804; and PCTPub. No. WO99/58723 and WO99/07865).

C.4. Sense Suppression

The SDFs of the reference, Sequence, Protein Group, and Protein GroupMatrix tables of the present invention are also useful to modulate geneexpression by sense suppression. Sense suppression represents anothermethod of gene suppression by introducing at least one exogenous copy orfragment of the endogenous sequence to be suppressed.

Introduction of expression cassettes in which a nucleic acid isconfigured in the sense orientation with respect to the promoter intothe chromosome of a plant or by a self-replicating virus has been shownto be an effective means by which to induce degradation of mRNAs oftarget genes. For an example of the use of this method to modulateexpression of endogenous genes see, Napoli et al., The Plant Cell 2:279(1990), and U.S. Pat. Nos. 5,034,323, 5,231,020, and 5,283,184.Inhibition of expression may require some transcription of theintroduced sequence.

For sense suppression, the introduced sequence generally will besubstantially identical to the endogenous sequence intended to beinactivated. The minimal percentage of sequence identity will typicallybe greater than about 65%, but a higher percentage of sequence identitymight exert a more effective reduction in the level of normal geneproducts. Sequence identity of more than about 80% is preferred, thoughabout 95% to absolute identity would be most preferred. As withantisense regulation, the effect would likely apply to any otherproteins within a similar family of genes exhibiting homology orsubstantial homology to the suppressing sequence.

C.5. Transcriptional Silencing

The nucleic acid sequences of the invention, including the SDFs of thereference, Sequence, Protein Group, and Protein Group Matrix tables, andfragments thereof, contain sequences that can be inserted into thegenome of an organism resulting in transcriptional silencing. Suchregulatory sequences need not be operatively linked to coding sequencesto modulate transcription of a gene. Specifically, a promoter sequencewithout any other element of a gene can be introduced into a genome totranscriptionally silence an endogenous gene (see, for example,Vaucheret, H et al. (1998) The Plant Journal 16: 651-659). As anotherexample, triple helices can be formed using oligonucleotides based onsequences from Reference, Sequence, Protein Group, and Protein GroupMatrix tables, fragments thereof, and substantially similar sequencethereto. The oligonucleotide can be delivered to the host cell and canbind to the promoter in the genome to form a triple helix and preventtranscription. An oligonucleotide of interest is one that can bind tothe promoter and block binding of a transcription factor to thepromoter. In such a case, the oligonucleotide can be complementary tothe sequences of the promoter that interact with transcription bindingfactors.

C.6. Other Methods to Inhibit Gene Expression

Yet another means of suppressing gene expression is to insert apolynucleotide into the gene of interest to disrupt transcription ortranslation of the gene.

Low frequency homologous recombination can be used to target apolynucleotide insert to a gene by flanking the polynucleotide insertwith sequences that are substantially similar to the gene to bedisrupted. Sequences from Reference, Sequence, Protein Group, andProtein Group Matrix tables, fragments thereof, and substantiallysimilar sequence thereto can be used for homologous recombination.

In addition, random insertion of polynucleotides into a host cell genomecan also be used to disrupt the gene of interest. Azpiroz-Leehan et al.,Trends in Genetics 13:152 (1997). In this method, screening for clonesfrom a library containing random insertions is preferred to identifyingthose that have polynucleotides inserted into the gene of interest. Suchscreening can be performed using probes and/or primers described abovebased on sequences from Reference, Sequence, Protein Group, and ProteinGroup Matrix tables, fragments thereof, and substantially similarsequence thereto. The screening can also be performed by selectingclones or R₁ plants having a desired phenotype.

I.D. Methods of Functional Analysis

The constructs described in the methods under I.C. above can be used todetermine the function of the polypeptide encoded by the gene that istargeted by the constructs.

Down-regulating the transcription and translation of the targeted genein the host cell or organisms, such as a plant, may produce phenotypicchanges as compared to a wild-type cell or organism. In addition, invitro assays can be used to determine if any biological activity, suchas calcium flux, DNA transcription, nucleotide incorporation, etc., arebeing modulated by the down-regulation of the targeted gene.

Coordinated regulation of sets of genes, e.g., those contributing to adesired polygenic trait, is sometimes necessary to obtain a desiredphenotype. SDFs of the invention representing transcription activationand DNA binding domains can be assembled into hybrid transcriptionalactivators. These hybrid transcriptional activators can be used withtheir corresponding DNA elements (i.e., those bound by the DNA-bindingSDFs) to effect coordinated expression of desired genes (J. J. Schwarzet al., Mol. Cell. Biol. 12:266 (1992), A. Martinez et al., Mol. Gen.Genet. 261:546 (1999)).

The SDFs of the invention can also be used in the two-hybrid geneticsystems to identify networks of protein-protein interactions (L.McAlister-Henn et al., Methods 19:330 (1999), J. C. Hu et al., Methods20:80 (2000), M. Golovkin et al., J. Biol. Chem. 274:36428 (1999), K.Ichimura et al., Biochem. Biophys. Res. Comm. 253:532 (1998)). The SDFsof the invention can also be used in various expression display methodsto identify important protein-DNA interactions (e.g. B. Luo et al., J.Mol. Biol. 266:479 (1997)).

I.E. Promoters

The SDFs of the invention are also useful as structural or regulatorysequences in a construct for modulating the expression of thecorresponding gene in a plant or other organism, e.g. a symbioticbacterium. For example, promoter sequences associated to SDFs of thereference, Sequence, Protein Group, and Protein Group Matrix tables ofthe present invention can be useful in directing expression of codingsequences either as constitutive promoters or to direct expression inparticular cell types, tissues, or organs or in response toenvironmental stimuli.

With respect to the SDFs of the present invention a promoter is likelyto be a relatively small portion of a genomic DNA (gDNA) sequencelocated in the first 2000 nucleotides upstream from an initial exonidentified in a gDNA sequence or initial “ATG” or methionine codon ortranslational start site in a corresponding cDNA sequence. Suchpromoters are more likely to be found in the first 1000 nucleotidesupstream of an initial ATG or methionine codon or translational startsite of a cDNA sequence corresponding to a gDNA sequence. In particular,the promoter is usually located upstream of the transcription startsite. The fragments of a particular gDNA sequence that function aselements of a promoter in a plant cell will preferably be found tohybridize to gDNA sequences presented and described in the Referencetable at medium or high stringency, relevant to the length of the probeand its base composition.

Promoters are generally modular in nature. Promoters can consist of abasal promoter that functions as a site for assembly of a transcriptioncomplex comprising an RNA polymerase, for example RNA polymerase II. Atypical transcription complex will include additional factors such asTF_(II)B, TF_(II)D, and TF_(II)E. Of these, TF_(II)D appears to be theonly one to bind DNA directly. The promoter might also contain one ormore enhancers and/or suppressors that function as binding sites foradditional transcription factors that have the function of modulatingthe level of transcription with respect to tissue specificity and oftranscriptional responses to particular environmental or nutritionalfactors, and the like.

Short DNA sequences representing binding sites for proteins can beseparated from each other by intervening sequences of varying length.For example, within a particular functional module, protein bindingsites may be constituted by regions of 5 to 60, preferably 10 to 30,more preferably 10 to 20 nucleotides. Within such binding sites, thereare typically 2 to 6 nucleotides that specifically contact amino acidsof the nucleic acid binding protein. The protein binding sites areusually separated from each other by 10 to several hundred nucleotides,typically by 15 to 150 nucleotides, often by 20 to 50 nucleotides. DNAbinding sites in promoter elements often display dyad symmetry in theirsequence. Often elements binding several different proteins, and/or aplurality of sites that bind the same protein, will be combined in aregion of 50 to 1,000 basepairs.

Elements that have transcription regulatory function can be isolatedfrom their corresponding endogenous gene, or the desired sequence can besynthesized, and recombined in constructs to direct expression of acoding region of a gene in a desired tissue-specific, temporal-specificor other desired manner of inducibility or suppression. Whenhybridizations are performed to identify or isolate elements of apromoter by hybridization to the long sequences presented in theReference tables, conditions are adjusted to account for theabove-described nature of promoters. For example short probes,constituting the element sought, are preferably used under lowtemperature and/or high salt conditions. When long probes, which mightinclude several promoter elements are used, low to medium stringencyconditions are preferred when hybridizing to promoters across species.

If a nucleotide sequence of an SDF, or part of the SDF, functions as apromoter or fragment of a promoter, then nucleotide substitutions,insertions or deletions that do not substantially affect the binding ofrelevant DNA binding proteins would be considered equivalent to theexemplified nucleotide sequence. It is envisioned that there areinstances where it is desirable to decrease the binding of relevant DNAbinding proteins to silence or down-regulate a promoter, or converselyto increase the binding of relevant. DNA binding proteins to enhance orup-regulate a promoter and vice versa. In such instances,polynucleotides representing changes to the nucleotide sequence of theDNA-protein contact region by insertion of additional nucleotides,changes to identity of relevant nucleotides, including use ofchemically-modified bases, or deletion of one or more nucleotides areconsidered encompassed by the present invention. In addition, fragmentsof the promoter sequences described by Reference tables and variantsthereof can be fused with other promoters or fragments to facilitatetranscription and/or transcription in specific type of cells or underspecific conditions.

Promoter function can be assayed by methods known in the art, preferablyby measuring activity of a reporter gene operatively linked to thesequence being tested for promoter function. Examples of reporter genesinclude those encoding luciferase, green fluorescent protein, GUS, neo,cat and bar.

I.F. UTRs and Junctions

Polynucleotides comprising untranslated (UTR) sequences and intron/exonjunctions are also within the scope of the invention. UTR sequencesinclude introns and 5′ or 3′ untranslated regions (5′ UTRs or 3′ UTRs).Fragments of the sequences shown in the Reference and Sequence tablescan comprise UTRs and intron/exon junctions.

These fragments of SDFs, especially UTRs, can have regulatory functionsrelated to, for example, translation rate and mRNA stability. Thus,these fragments of SDFs can be isolated for use as elements of geneconstructs for regulated production of polynucleotides encoding desiredpolypeptides.

Introns of genomic DNA segments might also have regulatory functions.Sometimes regulatory elements, especially transcription enhancer orsuppressor elements, are found within introns. Also, elements related tostability of heteronuclear RNA and efficiency of splicing and oftransport to the cytoplasm for translation can be found in intronelements. Thus, these segments can also find use as elements ofexpression vectors intended for use to transform plants.

Just as with promoters UTR sequences and intron/exon junctions can varyfrom those shown in the Reference and Sequence tables. Such changes fromthose sequences preferably will not affect the regulatory activity ofthe UTRs or intron/exon junction sequences on expression, transcription,or translation unless selected to do so. However, in some instances,down- or up-regulation of such activity may be desired to modulatetraits or phenotypic or in vitro activity.

I.G. Coding Sequences

Isolated polynucleotides of the invention can include coding sequencesthat encode polypeptides comprising an amino acid sequence encoded bysequences described in the Reference and Sequence tables or an aminoacid sequence presented in the Reference, Sequence, Protein Group, andProtein Group Matrix tables.

A nucleotide sequence encodes a polypeptide if a cell (or a cell free invitro system) expressing that nucleotide sequence produces a polypeptidehaving the recited amino acid sequence when the nucleotide sequence istranscribed and the primary transcript is subsequently processed andtranslated by a host cell (or a cell free in vitro system) harboring thenucleic acid. Thus, an isolated nucleic acid that encodes a particularamino acid sequence can be a genomic sequence comprising exons andintrons or a cDNA sequence that represents the product of splicingthereof. An isolated nucleic acid encoding an amino acid sequence alsoencompasses heteronuclear RNA, which contains sequences that are splicedout during expression, and mRNA, which lacks those sequences.

Coding sequences can be constructed using chemical synthesis techniquesor by isolating coding sequences or by modifying such synthesized orisolated coding sequences as described above.

In addition to coding sequences encoding the polypeptide sequences ofthe reference, Sequence, Protein Group, and Protein Group Matrix tables,which are native to corn, Arabidopsis, soybean, rice, wheat, and otherplants, the isolated polynucleotides can be polynucleotides that encodevariants, fragments, and fusions of those native proteins. Suchpolypeptides are described below in part II.

In variant polynucleotides generally, the number of substitutions,deletions or insertions is preferably less than 20%, more preferablyless than 15%; even more preferably less than 10%, 5%, 3% or 1% of thenumber of nucleotides comprising a particularly exemplified sequence. Itis generally expected that non-degenerate nucleotide sequence changesthat result in 1 to 10, more preferably 1 to 5 and most preferably 1 to3 amino acid insertions, deletions or substitutions will not greatlyaffect the function of an encoded polypeptide. The most preferredembodiments are those wherein 1 to 20, preferably 1 to 10, mostpreferably 1 to 5 nucleotides are added to, or deleted from and/orsubstituted in the sequences specifically disclosed in the Reference andSequence tables or polynucleotides that encode polypeptides of theProtein Group, and Protein Group Matrix tables or fragments thereof.

Insertions or deletions in polynucleotides intended to be used forencoding a polypeptide preferably preserve the reading frame. Thisconsideration is not so important in instances when the polynucleotideis intended to be used as a hybridization probe.

II. Polypeptides and Proteins

IIA. Native Polypeptides and Proteins

Polypeptides within the scope of the invention include both nativeproteins as well as variants, fragments, and fusions thereof.Polypeptides of the invention are those encoded by any of the sixreading frames of sequences shown in the Reference and Sequence tables,preferably encoded by the three frames reading in the 5′ to 3′ directionof the sequences as shown.

Native polypeptides include the proteins encoded by the sequences shownin the Reference and Sequence tables. Such native polypeptides includethose encoded by allelic variants.

Polypeptide and protein variants will exhibit at least 75% sequenceidentity to those native polypeptides of the Reference and Sequencetables. More preferably, the polypeptide variants will exhibit at least85% sequence identity; even more preferably, at least 90% sequenceidentity; more preferably at least 95%, 96%, 97%, 98%, or 99% sequenceidentity. Fragments of polypeptide or fragments of polypeptides willexhibit similar percentages of sequence identity to the relevantfragments of the native polypeptide. Fusions will exhibit a similarpercentage of sequence identity in that fragment of the fusionrepresented by the variant of the native peptide.

Polypeptide and protein variants of the invention will exhibit at least75% sequence identity to those motifs or consensus sequences of theProtein Group and Protein Group Matrix tables. More preferably, thepolypeptide variants will exhibit at least 85% sequence identity; evenmore preferably, at least 90% sequence identity; more preferably atleast 95%, 96%, 97%, 98%, or 99% sequence identity. Fragments ofpolypeptide or fragments of polypeptides will exhibit similarpercentages of sequence identity to the relevant fragments of the nativepolypeptide that are indicated in the Protein Group table. Fusions willexhibit a similar percentage of sequence identity in that fragment ofthe fusion represented by the variant of the native peptide.

Furthermore, polypeptide variants will exhibit at least one of thefunctional properties of the native protein. Such properties include,without limitation, protein interaction, DNA interaction, biologicalactivity, immunological activity, receptor binding, signal transduction,transcription activity, growth factor activity, secondary structure,three-dimensional structure, etc. As to properties related to in vitroor in vivo activities, the variants preferably exhibit at least 60% ofthe activity of the native protein; more preferably at least 70%, evenmore preferably at least 80%, 85%, 90% or 95% of at least one activityof the native protein.

One type of variant of native polypeptides comprises amino acidsubstitutions, deletions and/or insertions. Conservative substitutionsare preferred to maintain the function or activity of the polypeptide.

Within the scope of percentage of sequence identity described above, apolypeptide of the invention may have additional individual amino acidsor amino acid sequences inserted into the polypeptide in the middlethereof and/or at the N-terminal and/or C-terminal ends thereof.Likewise, some of the amino acids or amino acid sequences may be deletedfrom the polypeptide.

A.1 Antibodies

Isolated polypeptides can be utilized to produce antibodies.Polypeptides of the invention can generally be used, for example, asantigens for raising antibodies by known techniques. The resultingantibodies are useful as reagents for determining the distribution ofthe antigen protein within the tissues of a plant or within a cell of aplant. The antibodies are also useful for examining the production levelof proteins in various tissues, for example in a wild-type plant orfollowing genetic manipulation of a plant, by methods such as Westernblotting.

Antibodies of the present invention, both polyclonal and monoclonal, maybe prepared by conventional methods. In general, the polypeptides of theinvention are first used to immunize a suitable animal, such as a mouse,rat, rabbit, or goat. Rabbits and goats are preferred for thepreparation of polyclonal sera due to the volume of serum obtainable,and the availability of labeled anti-rabbit and anti-goat antibodies asdetection reagents. Immunization is generally performed by mixing oremulsifying the protein in saline, preferably in an adjuvant such asFreund's complete adjuvant, and injecting the mixture or emulsionparenterally (generally subcutaneously or intramuscularly). A dose of50-200 μg/injection is typically sufficient. Immunization is generallyboosted 2-6 weeks later with one or more injections of the protein insaline, preferably using Freund's incomplete adjuvant. One mayalternatively generate antibodies by in vitro immunization using methodsknown in the art, which for the purposes of this invention is consideredequivalent to in vivo immunization.

Polyclonal antisera is obtained by bleeding the immunized animal into aglass or plastic container, incubating the blood at 25° C. for one hour,followed by incubating the blood at 4° C. for 2-18 hours. The serum isrecovered by centrifugation (e.g., 1,000×g for 10 minutes). About 20-50ml per bleed may be obtained from rabbits.

Monoclonal antibodies are prepared using the method of Kohler andMilstein, Nature 256: 495 (1975), or modification thereof. Typically, amouse or rat is immunized as described above. However, rather thanbleeding the animal to extract serum, the spleen (and optionally severallarge lymph nodes) is removed and dissociated into single cells. Ifdesired, the spleen cells can be screened (after removal ofnonspecifically adherent cells) by applying a cell suspension to aplate, or well, coated with the protein antigen. B-cells producingmembrane-bound immunoglobulin specific for the antigen bind to theplate, and are not rinsed away with the rest of the suspension.Resulting B-cells, or all dissociated spleen cells, are then induced tofuse with myeloma cells to form hybridomas, and are cultured in aselective medium (e.g., hypoxanthine, aminopterin, thymidine medium,“HAT”). The resulting hybridomas are plated by limiting dilution, andare assayed for the production of antibodies which bind specifically tothe immunizing antigen (and which do not bind to unrelated antigens).The selected Mab-secreting hybridomas are then cultured either in vitro(e.g., in tissue culture bottles or hollow fiber reactors), or in vivo(as ascites in mice).

Other methods for sustaining antibody-producing B-cell clones, such asby EBV transformation, are known.

If desired, the antibodies (whether polyclonal or monoclonal) may belabeled using conventional techniques. Suitable labels includefluorophores, chromophores, radioactive atoms (particularly ³²P and¹²⁵I), electron-dense reagents, enzymes, and ligands having specificbinding partners. Enzymes are typically detected by their activity. Forexample, horseradish peroxidase is usually detected by its ability toconvert 3,3′,5,5′-tetramethylbenzidine (TNB) to a blue pigment,quantifiable with a spectrophotometer.

A.2 In Vitro Applications of Polypeptides

Some polypeptides of the invention will have enzymatic activities thatare useful in vitro. For example, the soybean trypsin inhibitor (Kunitz)family is one of the numerous families of proteinase inhibitors. Itcomprises plant proteins which have inhibitory activity against serineproteinases from the trypsin and subtilisin families, thiol proteinasesand aspartic proteinases. Thus, these peptides find in vitro use inprotein purification protocols and perhaps in therapeutic settingsrequiring topical application of protease inhibitors.

Delta-aminolevulinic acid dehydratase (EC 4.2.1.24) (ALAD) catalyzes thesecond step in the biosynthesis of heme, the condensation of twomolecules of 5-aminolevulinate to form porphobilinogen and is alsoinvolved in chlorophyll biosynthesis (Kaczor et al. (1994) PlantPhysiol. 1-4: 1411-7; Smith (1988) Biochem. J. 249: 423-8; Schneider(1976) Z. naturforsch. [C] 31: 55-63). Thus, ALAD proteins can be usedas catalysts in synthesis of heme derivatives. Enzymes of biosyntheticpathways generally can be used as catalysts for in vitro synthesis ofthe compounds representing products of the pathway.

Polypeptides encoded by SDFs of the invention can be engineered toprovide purification reagents to identify and purify additionalpolypeptides that bind to them. This allows one to identify proteinsthat function as multimers or elucidate signal transduction or metabolicpathways. In the case of DNA binding proteins, the polypeptide can beused in a similar manner to identify the DNA determinants of specificbinding (S. Pierrou et al., Anal. Biochem. 229:99 (1995), S.Chusacultanachai et al., J. Biol. Chem. 274:23591 (1999), Q. Lin et al.,J. Biol. Chem. 272:27274 (1997)).

II.B. Polypeptide Variants, Fragments and Fusions

Generally, variants, fragments, or fusions of the polypeptides encodedby the maximum length sequence (MLS) can exhibit at least one of theactivities of the identified domains and/or related polypeptidesdescribed in Sections (C) and (D) of The Reference tables correspondingto the MLS of interest.

II.B.(1) Variants

A type of variant of the native polypeptides comprises amino acidsubstitutions. Conservative substitutions, described above (see II.),are preferred to maintain the function or activity of the polypeptide.Such substitutions include conservation of charge, polarity,hydrophobicity, size, etc. For example, one or more amino acid residueswithin the sequence can be substituted with another amino acid ofsimilar polarity that acts as a functional equivalent, for exampleproviding a hydrogen bond in an enzymatic catalysis. Substitutes for anamino acid within an exemplified sequence are preferably made among themembers of the class to which the amino acid belongs. For example, thenonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine,valine, proline, phenylalanine, tryptophan and methionine. The polarneutral amino acids include glycine, serine, threonine, cysteine,tyrosine, asparagine, and glutamine. The positively charged (basic)amino acids include arginine, lysine and histidine. The negativelycharged (acidic) amino acids include aspartic acid and glutamic acid.

Within the scope of percentage of sequence identity described above, apolypeptide of the invention may have additional individual amino acidsor amino acid sequences inserted into the polypeptide in the middlethereof and/or at the N-terminal and/or C-terminal ends thereof.Likewise, some of the amino acids or amino acid sequences may be deletedfrom the polypeptide. Amino acid substitutions may also be made in thesequences; conservative substitutions being preferred.

One preferred class of variants are those that comprise (1) the domainof an encoded polypeptide and/or (2) residues conserved between theencoded polypeptide and related polypeptides. For this class ofvariants, the encoded polypeptide sequence is changed by insertion,deletion, or substitution at positions flanking the domain and/orconserved residues.

Another class of variants includes those that comprise an encodedpolypeptide sequence that is changed in the domain or conserved residuesby a conservative substitution.

Yet another class of variants includes those that lack one of the invitro activities, or structural features of the encoded polypeptides.One example is polypeptides or proteins produced from genes comprisingdominant negative mutations. Such a variant may comprise an encodedpolypeptide sequence with non-conservative changes in a particulardomain or group of conserved residues.

II.A.(2) Fragments

Fragments of particular interest are those that comprise a domainidentified for a polypeptide encoded by an MLS of the instant inventionand variants thereof. Also, fragments that comprise at least one regionof residues conserved between an MLS encoded polypeptide and its relatedpolypeptides are of great interest. Fragments are sometimes useful aspolypeptides corresponding to genes comprising dominant negativemutations are.

II.A.(3) Fusions

Of interest are chimeras comprising (1) a fragment of the MLS encodedpolypeptide or variants thereof of interest and (2) a fragment of apolypeptide comprising the same domain. For example, an AP2 helixencoded by a MLS of the invention fused to second AP2 helix from ANTprotein, which comprises two AP2 helices. The present invention alsoencompasses fusions of MLS encoded polypeptides, variants, or fragmentsthereof fused with related proteins or fragments thereof.

Definition of Domains

The polypeptides of the invention may possess identifying domains asshown in The Reference tables. Specific domains within the MLS encodedpolypeptides are indicated in The Reference tables. In addition, thedomains within the MLS encoded polypeptide can be defined by the regionthat exhibits at least 70% sequence identity with the consensussequences listed in the detailed description below of each of thedomains.

The majority of the protein domain descriptions given in the proteindomain table are obtained from Prosite (available on the internet), andPfam (also available on the internet). Examples of domain descriptionsare listed in the Protein Domain table.

A. Activities of Polypeptides Comprising Signal Peptides

Polypeptides comprising signal peptides are a family of proteins thatare typically targeted to (1) a particular organelle or intracellularcompartment, (2) interact with a particular molecule or (3) forsecretion outside of a host cell. Example of polypeptides comprisingsignal peptides include, without limitation, secreted proteins, solubleproteins, receptors, proteins retained in the ER, etc.

These proteins comprising signal peptides are useful to modulateligand-receptor interactions, cell-to-cell communication, signaltransduction, intracellular communication, and activities and/orchemical cascades that take part in an organism outside or within of anyparticular cell.

One class of such proteins are soluble proteins which are transportedout of the cell. These proteins can act as ligands that bind to receptorto trigger signal transduction or to permit communication between cells.

Another class is receptor proteins which also comprise a retentiondomain that lodges the receptor protein in the membrane when the celltransports the receptor to the surface of the cell. Like the solubleligands, receptors can also modulate signal transduction andcommunication between cells.

In addition the signal peptide itself can serve as a ligand for somereceptors. An example is the interaction of the ER targeting signalpeptide with the signal recognition particle (SRP). Here, the SRP bindsto the signal peptide, halting translation, and the resulting SRPcomplex then binds to docking proteins located on the surface of the ER,prompting transfer of the protein into the ER.

A description of signal peptide residue composition is described belowin Subsection IV.C.1.

III. Methods of Modulating Polypeptide Production

It is contemplated that polynucleotides of the invention can beincorporated into a host cell or in-vitro system to modulate polypeptideproduction. For instance, the SDFs prepared as described herein can beused to prepare expression cassettes useful in a number of techniquesfor suppressing or enhancing expression.

An example are polynucleotides comprising sequences to be transcribed,such as coding sequences, of the present invention can be inserted intonucleic acid constructs to modulate polypeptide production. Typically,such sequences to be transcribed are heterologous to at least oneelement of the nucleic acid construct to generate a chimeric gene orconstruct.

Another example of useful polynucleotides are nucleic acid moleculescomprising regulatory sequences of the present invention. Chimeric genesor constructs can be generated when the regulatory sequences of theinvention linked to heterologous sequences in a vector construct. Withinthe scope of invention are such chimeric gene and/or constructs.

Also within the scope of the invention are nucleic acid molecules,whereof at least a part or fragment of these DNA molecules are presentedin the Reference and Sequence tables or polynucleotide encodingpolypeptides of the Protein Group or Protein Group Matrix tables of thepresent application, and wherein the coding sequence is under thecontrol of its own promoter and/or its own regulatory elements. Suchmolecules are useful for transforming the genome of a host cell or anorganism regenerated from said host cell for modulating polypeptideproduction.

Additionally, a vector capable of producing the oligonucleotide can beinserted into the host cell to deliver the oligonucleotide.

More detailed description of components to be included in vectorconstructs are described both above and below.

Whether the chimeric vectors or native nucleic acids are utilized, suchpolynucleotides can be incorporated into a host cell to modulatepolypeptide production. Native genes and/or nucleic acid molecules canbe effective when exogenous to the host cell.

Methods of modulating polypeptide expression includes, withoutlimitation:

Suppression methods, such as

-   -   Antisense    -   Ribozymes    -   Co-suppression    -   Insertion of Sequences into the Gene to be Modulated    -   Regulatory Sequence Modulation.

as well as Methods for Enhancing Production, such as

-   -   Insertion of Exogenous Sequences; and    -   Regulatory Sequence Modulation.

III.A. Suppression

Expression cassettes of the invention can be used to suppress expressionof endogenous genes which comprise the SDF sequence. Inhibitingexpression can be useful, for instance, to tailor the ripeningcharacteristics of a fruit (Oeller et al., Science 254:437 (1991)) or toinfluence seed size (WO98/07842) or to provoke cell ablation (Mariani etal., Nature 357: 384-387 (1992).

As described above, a number of methods can be used to inhibit geneexpression in plants, such as antisense, ribozyme, introduction ofexogenous genes into a host cell, insertion of a polynucleotide sequenceinto the coding sequence and/or the promoter of the endogenous gene ofinterest, and the like.

III.A.1. Antisense

An expression cassette as described above can be transformed into hostcell or plant to produce an antisense strand of RNA. For plant cells,antisense RNA inhibits gene expression by preventing the accumulation ofmRNA which encodes the enzyme of interest, see, e.g., Sheehy et al.,Proc. Nat. Acad. Sci. USA, 85:8805 (1988), and Hiatt et al., U.S. Pat.No. 4,801,340.

III.A.2. Ribozymes

Similarly, ribozyme constructs can be transformed into a plant to cleavemRNA and down-regulate translation.

III.A.3. Co-Suppression

Another method of suppression is by introducing an exogenous copy of thegene to be suppressed. Introduction of expression cassettes in which anucleic acid is configured in the sense orientation with respect to thepromoter has been shown to prevent the accumulation of mRNA. A detaileddescription of this method is described above.

III.A.4. Insertion of Sequences into the Gene to be Modulated

Yet another means of suppressing gene expression is to insert apolynucleotide into the gene of interest to disrupt transcription ortranslation of the gene.

Homologous recombination could be used to target a polynucleotide insertto a gene using the Cre-Lox system (A. C. Vergunst et al., Nucleic AcidsRes. 26:2729 (1998), A. C. Vergunst et al., Plant Mol. Biol. 38:393(1998), H. Albert et al., Plant J. 7:649 (1995)).

In addition, random insertion of polynucleotides into a host cell genomecan also be used to disrupt the gene of interest. Azpiroz-Leehan et al.,Trends in Genetics 13:152 (1997). In this method, screening for clonesfrom a library containing random insertions is preferred for identifyingthose that have polynucleotides inserted into the gene of interest. Suchscreening can be performed using probes and/or primers described abovebased on sequences from the Reference and Sequence tables orpolynucleotides encoding polypeptides of the Protein Group or ProteinGroup Matrix tables, fragments thereof, and substantially similarsequence thereto. The screening can also be performed by selectingclones or any transgenic plants having a desired phenotype.

III.A.5. Regulatory Sequence Modulation

The SDFs described in the Reference and Sequence tables orpolynucleotides encoding polypeptides of the Protein Group or ProteinGroup Matrix tables, and fragments thereof are examples of nucleotidesof the invention that contain regulatory sequences that can be used tosuppress or inactivate transcription and/or translation from a gene ofinterest as discussed in I.C.5.

III.A.6. Genes Comprising Dominant-Negative Mutations

When suppression of production of the endogenous, native protein isdesired it is often helpful to express a gene comprising a dominantnegative mutation. Production of protein variants produced from genescomprising dominant negative mutations is a useful tool for researchGenes comprising dominant negative mutations can produce a variantpolypeptide which is capable of competing with the native polypeptide,but which does not produce the native result. Consequently, overexpression of genes comprising these mutations can titrate out anundesired activity of the native protein. For example, The product froma gene comprising a dominant negative mutation of a receptor can be usedto constitutively activate or suppress a signal transduction cascade,allowing examination of the phenotype and thus the trait(s) controlledby that receptor and pathway. Alternatively, the protein arising fromthe gene comprising a dominant-negative mutation can be an inactiveenzyme still capable of binding to the same substrate as the nativeprotein and therefore competes with such native protein.

Products from genes comprising dominant-negative mutations can also actupon the native protein itself to prevent activity. For example, thenative protein may be active only as a homo-multimer or as one subunitof a hetero-multimer. Incorporation of an inactive subunit into themultimer with native subunit(s) can inhibit activity.

Thus, gene function can be modulated in host cells of interest byinsertion into these cells vector constructs comprising a genecomprising a dominant-negative mutation.

III.B. Enhanced Expression

Enhanced expression of a gene of interest in a host cell can beaccomplished by either (1) insertion of an exogenous gene; or (2)promoter modulation.

III.B.1. Insertion of an Exogenous Gene

Insertion of an expression construct encoding an exogenous gene canboost the number of gene copies expressed in a host cell.

Such expression constructs can comprise genes that either encode thenative protein that is of interest or that encode a variant thatexhibits enhanced activity as compared to the native protein. Such genesencoding proteins of interest can be constructed from the sequences fromthe Reference and Sequence tables or polynucleotides encodingpolypeptides of the Protein Group or Protein Group Matrix tables,fragments thereof, and substantially similar sequence thereto.

Such an exogenous gene can include either a constitutive promoterpermitting expression in any cell in a host organism or a promoter thatdirects transcription only in particular cells or times during a hostcell life cycle or in response to environmental stimuli.

III.B.2. Regulatory Sequence Modulation

The SDFs of the Reference and Sequence tables, and fragments thereof,contain regulatory sequences that can be used to enhance expression of agene of interest. For example, some of these sequences contain usefulenhancer elements. In some cases, duplication of enhancer elements orinsertion of exogenous enhancer elements will increase expression of adesired gene from a particular promoter. As other examples, all 11promoters require binding of a regulatory protein to be activated, whilesome promoters may need a protein that signals a promoter bindingprotein to expose a polymerase binding site. In either case,over-production of such proteins can be used to enhance expression of agene of interest by increasing the activation time of the promoter.

Such regulatory proteins are encoded by some of the sequences in theReference and Sequence tables or polynucleotides encoding polypeptidesof the Protein Group or Protein Group Matrix tables, fragments thereof,and substantially similar sequences thereto.

Coding sequences for these proteins can be constructed as describedabove.

IV. Gene Constructs and Vector Construction

To use isolated SDFs of the present invention or a combination of themor parts and/or mutants and/or fusions of said SDFs in the abovetechniques, recombinant DNA vectors which comprise said SDFs and aresuitable for transformation of cells, such as plant cells, are usuallyprepared. The SDF construct can be made using standard recombinant DNAtechniques (Sambrook et al. 1989) and can be introduced to the speciesof interest by Agrobacterium-mediated transformation or by other meansof transformation (e.g., particle gun bombardment) as referenced below.

The vector backbone can be any of those typical in the art such asplasmids, viruses, artificial chromosomes, BACs, YACs and PACs andvectors of the sort described by

(a) BAC: Shizuya et al., Proc. Natl. Acad. Sci. USA 89: 8794-8797(1992); Hamilton et al., Proc. Natl. Acad. Sci. USA 93: 9975-9979(1996);

(b) YAC: Burke et al., Science 236:806-812 (1987);

(c) PAC: Sternberg N. et al., Proc Natl Acad Sci USA. January;87(1):103-7 (1990);

(d) Bacteria-Yeast Shuttle Vectors: Bradshaw et al., Nucl Acids Res 23:4850-4856 (1995);

(e) Lambda Phage Vectors: Replacement Vector, e.g., Frischauf et al., J.Mol Biol 170: 827-842 (1983); or Insertion vector, e.g., Huynh et al.,In: Glover N M (ed) DNA Cloning: A practical Approach, Vol. 1 Oxford:IRL Press (1985);

(f) T-DNA gene fusion vectors: Walden et al., Mol Cell Biol 1: 175-194(1990); and

(g) Plasmid vectors: Sambrook et al., infra.

Typically, a vector will comprise the exogenous gene, which in its turncomprises an SDF of the present invention to be introduced into thegenome of a host cell, and which gene may be an antisense construct, aribozyme construct chimeraplast, or a coding sequence with any desiredtranscriptional and/or translational regulatory sequences, such aspromoters, UTRs, and 3′ end termination sequences. Vectors of theinvention can also include origins of replication, scaffold attachmentregions (SARs), markers, homologous sequences, introns, etc.

A DNA sequence coding for the desired polypeptide, for example a cDNAsequence encoding a full length protein, will preferably be combinedwith transcriptional and translational initiation regulatory sequenceswhich will direct the transcription of the sequence from the gene in theintended tissues of the transformed plant.

For example, for over-expression, a plant promoter fragment may beemployed that will direct transcription of the gene in all tissues of aregenerated plant. Alternatively, the plant promoter may directtranscription of an SDF of the invention in a specific tissue(tissue-specific promoters) or may be otherwise under more preciseenvironmental control (inducible promoters).

If proper polypeptide production is desired, a polyadenylation region atthe 3′-end of the coding region is typically included. Thepolyadenylation region can be derived from the natural gene, from avariety of other plant genes, or from T-DNA.

The vector comprising the sequences from genes or SDF or the inventionmay comprise a marker gene that confers a selectable phenotype on plantcells. The vector can include promoter and coding sequence, forinstance. For example, the marker may encode biocide resistance,particularly antibiotic resistance, such as resistance to kanamycin,G418, bleomycin, hygromycin, or herbicide resistance, such as resistanceto chlorosulfuron or phosphinotricin.

IV.A. Coding Sequences

Generally, the sequence in the transformation vector and to beintroduced into the genome of the host cell does not need to beabsolutely identical to an SDF of the present invention. Also, it is notnecessary for it to be full length, relative to either the primarytranscription product or fully processed mRNA. Furthermore, theintroduced sequence need not have the same intron or exon pattern as anative gene. Also, heterologous non-coding segments can be incorporatedinto the coding sequence without changing the desired amino acidsequence of the polypeptide to be produced.

IV.B. Promoters

As explained above, introducing an exogenous SDF from the same speciesor an orthologous SDF from another species are useful to modulate theexpression of a native gene corresponding to that SDF of interest. Suchan SDF construct can be under the control of either a constitutivepromoter or a highly regulated inducible promoter (e.g., a copperinducible promoter). The promoter of interest can initially be eitherendogenous or heterologous to the species in question. Whenre-introduced into the genome of said species, such promoter becomesexogenous to said species. Over-expression of an SDF transgene can leadto co-suppression of the homologous endogeneous sequence therebycreating some alterations in the phenotypes of the transformed speciesas demonstrated by similar analysis of the chalcone synthase gene(Napoli et al., Plant Cell 2:279 (1990) and van der Krol et al., PlantCell 2:291 (1990)). If an SDF is found to encode a protein withdesirable characteristics, its over-production can be controlled so thatits accumulation can be manipulated in an organ- or tissue-specificmanner utilizing a promoter having such specificity.

Likewise, if the promoter of an SDF (or an SDF that includes a promoter)is found to be tissue-specific or developmentally regulated, such apromoter can be utilized to drive or facilitate the transcription of aspecific gene of interest (e.g., seed storage protein or root-specificprotein). Thus, the level of accumulation of a particular protein can bemanipulated or its spatial localization in an organ- or tissue-specificmanner can be altered.

IV.C Signal Peptides

SDFs of the present invention containing signal peptides are indicatedin the Reference, Sequence, the Protein Group and Protein Group Matrixtables. In some cases it may be desirable for the protein encoded by anintroduced exogenous or orthologous SDF to be targeted (1) to aparticular organelle intracellular compartment, (2) to interact with aparticular molecule such as a membrane molecule or (3) for secretionoutside of the cell harboring the introduced SDF. This will beaccomplished using a signal peptide.

Signal peptides direct protein targeting, are involved inligand-receptor interactions and act in cell to cell communication. Manyproteins, especially soluble proteins, contain a signal peptide thattargets the protein to one of several different intracellularcompartments. In plants, these compartments include, but are not limitedto, the endoplasmic reticulum (ER), mitochondria, plastids (such aschloroplasts), the vacuole, the Golgi apparatus, protein storagevessicles (PSV) and, in general, membranes. Some signal peptidesequences are conserved, such as the Asn-Pro-Ile-Arg amino acid motiffound in the N-terminal propeptide signal that targets proteins to thevacuole (Marty (1999) The Plant Cell 11: 587-599). Other signal peptidesdo not have a consensus sequence per se, but are largely composed ofhydrophobic amino acids, such as those signal peptides targetingproteins to the ER (Vitale and Denecke (1999) The Plant Cell 11:615-628). Still others do not appear to contain either a consensussequence or an identified common secondary sequence, for instance thechloroplast stromal targeting signal peptides (Keegstra and Cline (1999)The Plant Cell 11: 557-570). Furthermore, some targeting peptides arebipartite, directing proteins first to an organelle and then to amembrane within the organelle (e.g. within the thylakoid lumen of thechloroplast; see Keegstra and Cline (1999) The Plant Cell 11: 557-570).In addition to the diversity in sequence and secondary structure,placement of the signal peptide is also varied. Proteins destined forthe vacuole, for example, have targeting signal peptides found at theN-terminus, at the C-terminus and at a surface location in mature,folded proteins. Signal peptides also serve as ligands for somereceptors.

These characteristics of signal proteins can be used to more tightlycontrol the phenotypic expression of introduced SDFs. In particular,associating the appropriate signal sequence with a specific SDF canallow sequestering of the protein in specific organelles (plastids, asan example), secretion outside of the cell, targeting interaction withparticular receptors, etc. Hence, the inclusion of signal proteins inconstructs involving the SDFs of the invention increases the range ofmanipulation of SDF phenotypic expression. The nucleotide sequence ofthe signal peptide can be isolated from characterized genes using commonmolecular biological techniques or can be synthesized in vitro.

In addition, the native signal peptide sequences, both amino acid andnucleotide, described in the Reference, Sequence, Protein Group orProtein Group Matrix tables can be used to modulate polypeptidetransport. Further variants of the native signal peptides described inthe Reference, Sequence, Protein Group or Protein Group Matrix tablesare contemplated. Insertions, deletions, or substitutions can be made.Such variants will retain at least one of the functions of the nativesignal peptide as well as exhibiting some degree of sequence identity tothe native sequence.

Also, fragments of the signal peptides of the invention are useful andcan be fused with other signal peptides of interest to modulatetransport of a polypeptide.

V. Transformation Techniques

A wide range of techniques for inserting exogenous polynucleotides areknown for a number of host cells, including, without limitation,bacterial, yeast, mammalian, insect and plant cells.

Techniques for transforming a wide variety of higher plant species arewell known and described in the technical and scientific literature.See, e.g. Weising et al., Ann. Rev. Genet. 22:421 (1988); and Christou,Euphytica, v. 85, n. 1-3:13-27, (1995).

DNA constructs of the invention may be introduced into the genome of thedesired plant host by a variety of conventional techniques. For example,the DNA construct may be introduced directly into the genomic DNA of theplant cell using techniques such as electroporation and microinjectionof plant cell protoplasts, or the DNA constructs can be introduceddirectly to plant tissue using ballistic methods, such as DNA particlebombardment. Alternatively, the DNA constructs may be combined withsuitable T-DNA flanking regions and introduced into a conventionalAgrobacterium tumefaciens host vector. The virulence functions of theAgrobacterium tumefaciens host will direct the insertion of theconstruct and adjacent marker into the plant cell DNA when the cell isinfected by the bacteria (McCormac et al., Mol. Biotechnol. 8:199(1997); Hamilton, Gene 200:107 (1997)); Salomon et al. EMBO J. 3:141(1984); Herrera-Estrella et al. EMBO J. 2:987 (1983).

Microinjection techniques are known in the art and well described in thescientific and patent literature. The introduction of DNA constructsusing polyethylene glycol precipitation is described in Paszkowski etal. EMBO J. 3:2717 (1984). Electroporation techniques are described inFromm et al. Proc. Natl. Acad. Sci. USA 82:5824 (1985). Ballistictransformation techniques are described in Klein et al. Nature 327:773(1987). Agrobacterium tumefaciens-mediated transformation techniques,including disarming and use of binary or co-integrate vectors, are welldescribed in the scientific literature. See, for example Hamilton, C M.,Gene 200:107 (1997); Müller et al. Mol. Gen. Genet. 207:171 (1987);Komari et al. Plant J. 10:165 (1996); Venkateswarlu et al. Biotechnology9:1103 (1991) and Gleave, A P., Plant Mol. Biol. 20:1203 (1992); Gravesand Goldman, Plant Mol. Biol. 7:34 (1986) and Gould et al., PlantPhysiology 95:426 (1991).

Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantthat possesses the transformed genotype and thus the desired phenotypesuch as seedlessness. Such regeneration techniques rely on manipulationof certain phytohormones in a tissue culture growth medium, typicallyrelying on a biocide and/or herbicide marker which has been introducedtogether with the desired nucleotide sequences. Plant regeneration fromcultured protoplasts is described in Evans et al., Protoplasts Isolationand Culture in “Handbook of Plant Cell Culture,” pp. 124-176, MacMillanPublishing Company, New York, 1983; and Binding, Regeneration of Plants,Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1988. Regenerationcan also be obtained from plant callus, explants, organs, or partsthereof. Such regeneration techniques are described generally in Klee etal. Ann. Rev. of Plant Phys. 38:467 (1987). Regeneration of monocots(rice) is described by Hosoyama et al. (Biosci. Biotechnol. Biochem.58:1500 (1994)) and by Ghosh et al. (J. Biotechnol. 32:1 (1994)). Thenucleic acids of the invention can be used to confer desired traits onessentially any plant.

Thus, the invention has use over a broad range of plants, includingspecies from the genera Anacardium, Arachis, Asparagus, Atropa, Avena,Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea,Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium,Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum,Lolium,Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago,Nicotiana, Olea, Oryza, Panieum, Pannesetum, Persea, Phaseolus,Pistachia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio,Sinapis, Solanum, Sorghum, Theobromus, Trigonella, Triticum, Vicia,Vitis, Vigna, and, Zea.

One of skill will recognize that after the expression cassette is stablyincorporated in transgenic plants and confirmed to be operable, it canbe introduced into other plants by sexual crossing. Any of a number ofstandard breeding techniques can be used, depending upon the species tobe crossed.

The particular sequences of SDFs identified are provided in the attachedReference and Sequence tables.

IX. Definitions

The following terms are utilized throughout this application:

Allelic variant: An “allelic variant” is an alternative form of the sameSDF, which resides at the same chromosomal locus in the organism.Allelic variations can occur in any portion of the gene sequence,including regulatory regions. Allelic variants can arise by normalgenetic variation in a population. Allelic variants can also be producedby genetic engineering methods. An allelic variant can be one that isfound in a naturally occurring plant, including a cultivar or ecotype.An allelic variant may or may not give rise to a phenotypic change, andmay or may not be expressed. An allele can result in a detectable changein the phenotype of the trait represented by the locus. A phenotypicallysilent allele can give rise to a product.

Alternatively spliced messages: Within the context of the currentinvention, “alternatively spliced messages” refers to mature mRNAsoriginating from a single gene with variations in the number and/oridentity of exons, introns and/or intron-exon junctions.

Chimeric: The term “chimeric” is used to describe genes, as definedsupra, or contructs wherein at least two of the elements of the gene orconstruct, such as the promoter and the coding sequence and/or otherregulatory sequences and/or filler sequences and/or complements thereof,are heterologous to each other.

Constitutive Promoter: Promoters referred to herein as “constitutivepromoters” actively promote transcription under most, but notnecessarily all, environmental conditions and states of development orcell differentiation. Examples of constitutive promoters include thecauliflower mosaic virus (CaMV) 35S transcript initiation region and the1′ or 2′ promoter derived from T-DNA of Agrobacterium tumefaciens, andother transcription initiation regions from various plant genes, such asthe maize ubiquitin-1 promoter, known to those of skill.

Coordinately Expressed: The term “coordinately expressed,” as used inthe current invention, refers to genes that are expressed at the same ora similar time and/or stage and/or under the same or similarenvironmental conditions.

Domain: Domains are fingerprints or signatures that can be used tocharacterize protein families and/or parts of proteins. Suchfingerprints or signatures can comprise conserved (1) primary sequence,(2) secondary structure, and/or (3) three-dimensional conformation.Generally, each domain has been associated with either a family ofproteins or motifs. Typically, these families and/or motifs have beencorrelated with specific in-vitro and/or in-vivo activities. A domaincan be any length, including the entirety of the sequence of a protein.Detailed descriptions of the domains, associated families and motifs,and correlated activities of the polypeptides of the instant inventionare described below. Usually, the polypeptides with designated domain(s)can exhibit at least one activity that is exhibited by any polypeptidethat comprises the same domain(s).

Endogenous: The term “endogenous,” within the context of the currentinvention refers to any polynucleotide, polypeptide or protein sequencewhich is a natural part of a cell or organisms regenerated from saidcell.

Exogenous: “Exogenous,” as referred to within, is any polynucleotide,polypeptide or protein sequence, whether chimeric or not, that isinitially or subsequently introduced into the genome of an individualhost cell or the organism regenerated from said host cell by any meansother than by a sexual cross. Examples of means by which this can beaccomplished are described below, and include Agrobacterium-mediatedtransformation (of dicots—e.g. Salomon et al. EMBO J. 3:141 (1984);Herrera-Estrella et al. EMBO J. 2:987 (1983); of monocots,representative papers are those by Escudero et al., Plant J. 10:355(1996), Ishida et al., Nature Biotechnology 14:745 (1996), May et al.,Bio/Technology 13:486 (1995)), biolistic methods (Armaleo et al.,Current Genetics 17:97 1990)), electroporation, in planta techniques,and the like. Such a plant containing the exogenous nucleic acid isreferred to here as a T₀ for the primary transgenic plant and T₁ for thefirst generation. The term “exogenous” as used herein is also intendedto encompass inserting a naturally found element into a non-naturallyfound location.

Filler sequence: As used herein, “filler sequence” refers to anynucleotide sequence that is inserted into DNA construct to evoke aparticular spacing between particular components such as a promoter anda coding region and may provide an additional attribute such as arestriction enzyme site.

Gene: The term “gene,” as used in the context of the current invention,encompasses all regulatory and coding sequence contiguously associatedwith a single hereditary unit with a genetic function (see SCHEMATIC 1).Genes can include non-coding sequences that modulate the geneticfunction that include, but are not limited to, those that specifypolyadenylation, transcriptional regulation, DNA conformation, chromatinconformation, extent and position of base methylation and binding sitesof proteins that control all of these. Genes comprised of “exons”(coding sequences), which may be interrupted by “introns” (non-codingsequences), encode proteins. A gene's genetic function may require onlyRNA expression or protein production, or may only require binding ofproteins and/or nucleic acids without associated expression. In certaincases, genes adjacent to one another may share sequence in such a waythat one gene will overlap the other. A gene can be found within thegenome of an organism, artificial chromosome, plasmid, vector, etc., oras a separate isolated entity.

Gene Family: “Gene family” is used in the current invention to describea group of functionally related genes, each of which encodes a separateprotein.

Heterologous sequences: “Heterologous sequences” are those that are notoperatively linked or are not contiguous to each other in nature. Forexample, a promoter from corn is considered heterologous to anArabidopsis coding region sequence. Also, a promoter from a geneencoding a growth factor from corn is considered heterologous to asequence encoding the corn receptor for the growth factor. Regulatoryelement sequences, such as UTRs or 3′ end termination sequences that donot originate in nature from the same gene as the coding sequenceoriginates from, are considered heterologous to said coding sequence.Elements operatively linked in nature and—contiguous to each other arenot heterologous to each other. On the other hand, these same elementsremain operatively linked but become heterologous if other fillersequence is placed between them. Thus, the promoter and coding sequencesof a corn gene expressing an amino acid transporter are not heterologousto each other, but the promoter and coding sequence of a corn geneoperatively linked in a novel manner are heterologous.

Homologous gene: In the current invention, “homologous gene” refers to agene that shares sequence similarity with the gene of interest. Thissimilarity may be in only a fragment of the sequence and oftenrepresents a functional domain such as, examples including withoutlimitation a DNA binding domain, a domain with tyrosine kinase activity,or the like. The functional activities of homologous genes are notnecessarily the same.

Inducible Promoter: An “inducible promoter” in the context of thecurrent invention refers to a promoter which is regulated under certainconditions, such as light, chemical concentration, proteinconcentration, conditions in an organism, cell, or organelle, etc. Atypical example of an inducible promoter, which can be utilized with thepolynucleotides of the present invention, is PARSK1, the promoter fromthe Arabidopsis gene encoding a serine-threonine kinase enzyme, andwhich promoter is induced by dehydration, abscissic acid and sodiumchloride (Wang and Goodman, Plant J. 8:37 (1995)) Examples ofenvironmental conditions that may affect transcription by induciblepromoters include anaerobic conditions, elevated temperature, or thepresence of light.

Intergenic region: “Intergenic region,” as used in the currentinvention, refers to nucleotide sequence occurring in the genome thatseparates adjacent genes.

Mutant gene: In the current invention, “mutant” refers to a heritablechange in DNA sequence at a specific location. Mutants of the currentinvention may or may not have an associated identifiable function whenthe mutant gene is transcribed.

Orthologous Gene In the current invention “orthologous gene” refers to asecond gene that encodes a gene product that performs a similar functionas the product of a first gene. The orthologous gene may also have adegree of sequence similarity to the first gene. The orthologous genemay encode a polypeptide that exhibits a degree of sequence similarityto a polypeptide corresponding to a first gene. The sequence similaritycan be found within a functional domain or along the entire length ofthe coding sequence of the genes and/or their correspondingpolypeptides.

Percentage of sequence identity: “Percentage of sequence identity,” asused herein, is determined by comparing two optimally aligned sequencesover a comparison window, where the fragment of the polynucleotide oramino acid sequence in the comparison window may comprise additions ordeletions (e.g., gaps or overhangs) as compared to the referencesequence (which does not comprise additions or deletions) for optimalalignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity. Optimalalignment of sequences for comparison may be conducted by the localhomology algorithm of Smith and Waterman Add. APL. Math. 2:482 (1981),by the homology alignment algorithm of Needleman and Wunsch J. Mol.Biol. 48:443 (1970), by the search for similarity method of Pearson andLipman Proc. Natl. Acad. Sci. (USA) 85: 2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, BLAST, PASTA, andTFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup (GCG), 575 Science Dr., Madison, Wis.), or by inspection. Giventhat two sequences have been identified for comparison, GAP and BESTFITare preferably employed to determine their optimal alignment. Typically,the default values of 5.00 for gap weight and 0.30 for gap weight lengthare used. The term “substantial sequence identity” betweenpolynucleotide or polypeptide sequences refers to polynucleotide orpolypeptide comprising a sequence that has at least 80% sequenceidentity, preferably at least 85%, more preferably at least 90% and mostpreferably at least 95%, even more preferably, at least 96%, 97%, 98% or99% sequence identity compared to a reference sequence using theprograms.

Plant Promoter: A “plant promoter” is a promoter capable of initiatingtranscription in plant cells and can drive or facilitate transcriptionof a fragment of the SDF of the instant invention or a coding sequenceof the SDF of the instant invention. Such promoters need not be of plantorigin. For example, promoters derived from plant viruses, such as theCaMV35S promoter or from Agrobacterium tumefaciens such as the T-DNApromoters, can be plant promoters. A typical example of a plant promoterof plant origin is the maize ubiquitin-1 (ubi-1) promoter known to thoseof skill.

Promoter: The term “promoter,” as used herein, refers to a region ofsequence determinants located upstream from the start of transcriptionof a gene and which are involved in recognition and binding of RNApolymerase and other proteins to initiate and modulate transcription. Abasal promoter is the minimal sequence necessary for assembly of atranscription complex required for transcription initiation. Basalpromoters frequently include a “TATA box” element usually locatedbetween 15 and 35 nucleotides upstream from the site of initiation oftranscription. Basal promoters also sometimes include a “CCAAT box”element (typically a sequence CCAAT) and/or a GGGCG sequence, usuallylocated between 40 and 200 nucleotides, preferably 60 to 120nucleotides, upstream from the start site of transcription.

Public sequence: The term “public sequence,” as used in the context ofthe instant application, refers to any sequence that has been depositedin a publicly accessible database. This term encompasses both amino acidand nucleotide sequences. Such sequences are publicly accessible, forexample, on the BLAST databases on the NCBI FTP web site (accessible atncbi.nlm.gov/blast). The database at the NCBI GTP site utilizes “gi”numbers assigned by NCBI as a unique identifier for each sequence in thedatabases, thereby providing a non-redundant database for sequence fromvarious databases, including GenBank, EMBL, DBBJ, (DNA Database ofJapan) and PDB (Brookhaven Protein Data Bank).

Regulatory Sequence The term “regulatory sequence,” as used in thecurrent invention, refers to any nucleotide sequence that influencestranscription or translation initiation and rate, and stability and/ormobility of the transcript or polypeptide product. Regulatory sequencesinclude, but are not limited to, promoters, promoter control elements,protein binding sequences, 5′ and 3′ UTRs, transcriptional start site,termination sequence, polyadenylation sequence, introns, certainsequences within a coding sequence, etc.

Related Sequences: “Related sequences” refer to either a polypeptide ora nucleotide sequence that exhibits some degree of sequence similaritywith a sequence described by The Reference tables and The Sequencetables.

Scaffold Attachment Region (SAR): As used herein, “scaffold attachmentregion” is a DNA sequence that anchors chromatin to the nuclear matrixor scaffold to generate loop domains that can have either atranscriptionally active or inactive structure (Spiker and Thompson(1996) Plant Physiol. 110: 15-21).

Sequence-determined DNA fragments (SDFs): “Sequence-determined DNAfragments” as used in the current invention are isolated sequences ofgenes, fragments of genes, intergenic regions or contiguous DNA fromplant genomic DNA or cDNA or RNA the sequence of which has beendetermined.

Signal Peptide: A “signal peptide” as used in the current invention isan amino acid sequence that targets the protein for secretion, fortransport to an intracellular compartment or organelle or forincorporation into a membrane. Signal peptides are indicated in thetables and a more detailed description located below.

Specific Promoter: In the context of the current invention, “specificpromoters” refers to a subset of inducible promoters that have a highpreference for being induced in a specific tissue or cell and/or at aspecific time during development of an organism. By “high preference” ismeant at least 3-fold, preferably 5-fold, more preferably at least10-fold still more preferably at least 20-fold, 50-fold or 100-foldincrease in transcription in the desired tissue over the transcriptionin any other tissue. Typical examples of temporal and/or tissue specificpromoters of plant origin that can be used with the polynucleotides ofthe present invention, are: PTA29, a promoter which is capable ofdriving gene transcription specifically in tapetum and only duringanther development (Koltonow et al., Plant Cell 2:1201 (1990); RCc2 andRCc3, promoters that direct root-specific gene transcription in rice (Xuet al., Plant Mol. Biol. 27:237 (1995); TobRB27, a root-specificpromoter from tobacco (Yamamoto et al., Plant Cell 3:371 (1991)).Examples of tissue-specific promoters under developmental controlinclude promoters that initiate transcription only in certain tissues ororgans, such as root, ovule, fruit, seeds, or flowers. Other suitablepromoters include those from genes encoding storage proteins or thelipid body membrane protein, oleosin. A few root-specific promoters arenoted above.

Stringency: “Stringency” as used herein is a function of probe length,probe composition (G+C content), and salt concentration, organic solventconcentration, and temperature of hybridization or wash conditions.Stringency is typically compared by the parameter T_(m), which is thetemperature at which 50% of the complementary molecules in thehybridization are hybridized, in terms of a temperature differentialfrom T_(m). High stringency conditions are those providing a conditionof T_(m)−5° C. to T_(m)−10° C. Medium or moderate stringency conditionsare those providing T_(m)−20° C. to T_(m)−29° C. Low stringencyconditions are those providing a condition of T_(m)−40° C. to T_(m)−48°C. The relationship of hybridization conditions to T_(m) (in ° C.) isexpressed in the mathematical equation

T _(m)=81.5−16.6(log₁₀[Na⁺])+0.41(% G+C)−(600/N)  (1)

where N is the length of the probe. This equation works well for probes14 to 70 nucleotides in length that are identical to the targetsequence. The equation below for T_(m) of DNA-DNA hybrids is useful forprobes in the range of 50 to greater than 500 nucleotides, and forconditions that include an organic solvent (formamide).

T _(m)=81.5+16.6 log {[Na⁺]/(1+0.7[Na⁺])}+0.41(% G+C)−500/L0.63(%formamide)  (2)

where L is the length of the probe in the hybrid. (P. Tijessen,“Hybridization with Nucleic Acid Probes” in Laboratory Techniques inBiochemistry and Molecular Biology, P. C. vand der Vliet, ed., c. 1993by Elsevier, Amsterdam.) The T_(m) of equation (2) is affected by thenature of the hybrid; for DNA-RNA hybrids T_(m) is 10-15° C. higher thancalculated, for RNA-RNA hybrids T_(m) is 20-25° C. higher. Because theT_(m) decreases about 1° C. for each 1% decrease in homology when a longprobe is used (Bonner et al., J. Mol. Biol. 81:123 (1973)), stringencyconditions can be adjusted to favor detection of identical genes orrelated family members.

Equation (2) is derived assuming equilibrium and therefore,hybridizations according to the present invention are most preferablyperformed under conditions of probe excess and for sufficient time toachieve equilibrium. The time required to reach equilibrium can beshortened by inclusion of a hybridization accelerator such as dextransulfate or another high volume polymer in the hybridization buffer.

Stringency can be controlled during the hybridization reaction or afterhybridization has occurred by altering the salt and temperatureconditions of the wash solutions used. The formulas shown above areequally valid when used to compute the stringency of a wash solution.Preferred wash solution stringencies lie within the ranges stated above;high stringency is 5-8° C. below T_(m), medium or moderate stringency is26-29° C. below T_(m) and low stringency is 45-48° C. below T_(m).

Substantially free of: A composition containing A is “substantially freeof” B when at least 85% by weight of the total A+B in the composition isA. Preferably, A comprises at least about 90% by weight of the total ofA+B in the composition, more preferably at least about 95% or even 99%by weight. For example, a plant gene or DNA sequence can be consideredsubstantially free of other plant genes or DNA sequences.

Translational start site: In the context of the current invention, a“translational start site” is usually an ATG in the cDNA transcript,more usually the first ATG. A single cDNA, however, may have multipletranslational start sites.

Transcription start site: “Transcription start site” is used in thecurrent invention to describe the point at which transcription isinitiated. This point is typically located about 25 nucleotidesdownstream from a TFIID binding site, such as a TATA box. Transcriptioncan initiate at one or more sites within the gene, and a single gene mayhave multiple transcriptional start sites, some of which may be specificfor transcription in a particular cell-type or tissue.

Untranslated region (UTR): A “UTR” is any contiguous series ofnucleotide bases that is transcribed, but is not translated. Theseuntranslated regions may be associated with particular functions such asincreasing mRNA message stability. Examples of UTRs include, but are notlimited to polyadenylation signals, terminations sequences, sequenceslocated between the transcriptional start site and the first exon (5′UTR) and sequences located between the last exon and the end of the mRNA(3′ UTR).

Variant: The term “variant” is used herein to denote a polypeptide orprotein or polynucleotide molecule that differs from others of its kindin some way. For example, polypeptide and protein variants can consistof changes in amino acid sequence and/or charge and/orpost-translational modifications (such as glycosylation, etc).

X. Examples

The invention is illustrated by way of the following examples. Theinvention is not limited by these examples as the scope of the inventionis defined solely by the claims following.

Example 1 cDNA Preparation

A number of the nucleotide sequences disclosed in the Reference andSequence tables or polynucleotides encoding polypeptides of the ProteinGroup or Protein Group Matrix tables, herein as representative of theSDFs of the invention can be obtained by sequencing genomic DNA (gDNA)and/or cDNA from corn plants grown from HYBRID SEED # 35A19, purchasedfrom Pioneer Hi-Bred International, Inc., Supply Management, P.O. Box256, Johnston, Iowa 50131-0256.

A number of the nucleotide sequences disclosed in the Reference andSequence tables or polynucleotides encoding polypeptides of the ProteinGroup or Protein Group Matrix tables, herein as representative of theSDFs of the invention can also be obtained by sequencing genomic DNAfrom Arabidopsis thaliana, Wassilewskija ecotype or by sequencing cDNAobtained from mRNA from such plants as described below. This is a truebreeding strain. Seeds of the plant are available from the ArabidopsisBiological Resource Center at the Ohio State University, under theaccession number CS2360. Seeds of this plant were deposited under theterms and conditions of the Budapest Treaty at the American Type CultureCollection, Manassas, Va. on Aug. 31, 1999, and were assigned ATCC No.PTA-595.

Other methods for cloning full-length cDNA are described, for example,by Seki et al., Plant Journal 15:707-720 (1998) “High-efficiency cloningof Arabidopsis full-length cDNA by biotinylated Cap trapper”; Maruyamaet al., Gene 138:171 (1994) “Oligo-capping a simple method to replacethe cap structure of eukaryotic mRNAs with oligoribonucleotides”; and WO96/34981.

Tissues were, or each organ was, individually pulverized and frozen inliquid nitrogen. Next, the samples were homogenized in the presence ofdetergents and then centrifuged. The debris and nuclei were removed fromthe sample and more detergents were added to the sample. The sample wascentrifuged and the debris was removed. Then the sample was applied to a2M sucrose cushion to isolate polysomes. The RNA was isolated bytreatment with detergents and proteinase K followed by ethanolprecipitation and centrifugation. The polysomal RNA from the differenttissues was pooled according to the following mass ratios: 15/15/1 formale inflorescences, female inflorescences and root, respectively. Thepooled material was then used for cDNA synthesis by the methodsdescribed below.

Starting material for cDNA synthesis for the exemplary corn cDNA cloneswith sequences presented in the Reference and Sequence tables orpolynucleotides encoding polypeptides of the Protein Group or ProteinGroup Matrix tables was poly(A)-containing polysomal mRNAs frominflorescences and root tissues of corn plants grown from HYBRID SEED #35A19. Male inflorescences and female (pre- and post-fertilization)inflorescences were isolated at various stages of development. Selectionfor poly(A) containing polysomal RNA was done using oligo d(T) cellulosecolumns, as described by Cox and Goldberg, “Plant Molecular Biology: APractical Approach”, pp. 1-35, Shaw ed., c. 1988 by IRL, Oxford. Thequality and the integrity of the polyA+ RNAs were evaluated.

Starting material for cDNA synthesis for the exemplary Arabidopsis cDNAclones with sequences presented in the Reference and Sequence tables orpolynucleotides encoding polypeptides of the Protein Group or ProteinGroup Matrix tables was polysomal RNA isolated from the top-mostinflorescence tissues of Arabidopsis thaliana Wassilewskija (Ws.) andfrom roots of Arabidopsis thaliana Landsberg erecta (L. er.), alsoobtained from the Arabidopsis Biological Resource Center. Nine partsinflorescence to every part root was used, as measured by wet mass.Tissue was pulverized and exposed to liquid nitrogen. Next, the samplewas homogenized in the presence of detergents and then centrifuged. Thedebris and nuclei were removed from the sample and more detergents wereadded to the sample. The sample was centrifuged and the debris wasremoved and the sample was applied to a 2M sucrose cushion to isolatepolysomal RNA. Cox et al., “Plant Molecular Biology: A PracticalApproach”, pp. 1-35, Shaw ed., c. 1988 by IRL, Oxford. The polysomal RNAwas used for cDNA synthesis by the methods described below. PolysomalmRNA was then isolated as described above for corn cDNA. The quality ofthe RNA was assessed electrophoretically.

Following preparation of the mRNAs from various tissues as describedabove, selection of mRNA with intact 5′ ends and specific attachment ofan oligonucleotide tag to the 5′ end of such mRNA was performed usingeither a chemical or enzymatic approach. Both techniques take advantageof the presence of the “cap” structure, which characterizes the 5′ endof most intact mRNAs and which comprises a guanosine generallymethylated once, at the 7 position.

The chemical modification approach involves the optional elimination ofthe 2′,3′-cis diol of the 3′ terminal ribose, the oxidation of the2′,3′-cis diol of the ribose linked to the cap of the 5′ ends of themRNAs into a dialdehyde, and the coupling of the such obtaineddialdehyde to a derivatized oligonucleotide tag. Further detailregarding the chemical approaches for obtaining mRNAs having intact 5′ends are disclosed in International Application No. WO96/34981 publishedNov. 7, 1996.

The enzymatic approach for ligating the oligonucleotide tag to theintact 5′ ends of mRNAs involves the removal of the phosphate groupspresent on the 5′ ends of uncapped incomplete mRNAs, the subsequentdecapping of mRNAs having intact 5′ ends and the ligation of thephosphate present at the 5′ end of the decapped mRNA to anoligonucleotide tag. Further detail regarding the enzymatic approachesfor obtaining mRNAs having intact 5′ ends are disclosed in Dumas MilneEdwards J. B. (Doctoral Thesis of Paris VI University, Le clonage desADNc complets: difficultes et perspectives nouvelles. Apports pourl'étude de la régulation de l'expression de la tryptophane hydroxylasede rat, 20 Dec. 1993), EPO 625572 and Kato et al., Gene 150:243-250(1994).

In both the chemical and the enzymatic approach, the oligonucleotide taghas a restriction enzyme site (e.g. an EcoRI site) therein to facilitatelater cloning procedures. Following attachment of the oligonucleotidetag to the mRNA, the integrity of the mRNA is examined by performing aNorthern blot using a probe complementary to the oligonucleotide tag.

For the mRNAs joined to oligonucleotide tags using either the chemicalor the enzymatic method, first strand cDNA synthesis is performed usingan oligo-dT primer with reverse transcriptase. This oligo-dT primer cancontain an internal tag of at least 4 nucleotides, which can bedifferent from one mRNA preparation to another. Methylated dCTP is usedfor cDNA first strand synthesis to protect the internal EcoRI sites fromdigestion during subsequent steps. The first strand cDNA is precipitatedusing isopropanol after removal of RNA by alkaline hydrolysis toeliminate residual primers.

Second strand cDNA synthesis is conducted using a DNA polymerase, suchas Klenow fragment and a primer corresponding to the 5′ end of theligated oligonucleotide. The primer is typically 20-25 bases in length.Methylated dCTP is used for second strand synthesis in order to protectinternal EcoRI sites in the cDNA from digestion during the cloningprocess.

Following second strand synthesis, the full-length cDNAs are cloned intoa phagemid vector, such as pBlueScript™ (Stratagene). The ends of thefull-length cDNAs are blunted with T4 DNA polymerase (Biolabs) and thecDNA is digested with EcoRI. Since methylated dCTP is used during cDNAsynthesis, the EcoRI site present in the tag is the only hemi-methylatedsite; hence the only site susceptible to EcoRI digestion. In someinstances, to facilitate subcloning, an Hind III adapter is added to the3 end of full-length cDNAs.

The full-length cDNAs are then size fractionated using either exclusionchromatography (AcA, Biosepra) or electrophoretic separation whichyields 3 to 6 different fractions. The full-length cDNAs are thendirectionally cloned either into pBlueScript™ using either the EcoRI andSmaI restriction sites or, when the Hind III adapter is present in thefull-length cDNAs, the EcoRI and Hind III restriction sites. Theligation mixture is transformed, preferably by electroporation, intobacteria, which are then propagated under appropriate antibioticselection.

Clones containing the oligonucleotide tag attached to full-length cDNAsare selected as follows.

The plasmid cDNA libraries made as described above are purified (e.g. bya column available from Qiagen). A positive selection of the taggedclones is performed as follows. Briefly, in this selection procedure,the plasmid DNA is converted to single stranded DNA using phage F1 geneII endonuclease in combination with an exonuclease (Chang et al., Gene127:95 (1993)) such as exonuclease III or T7 gene 6 exonuclease. Theresulting single stranded DNA is then purified using paramagnetic beadsas described by Fry et al., Biotechniques 13: 124 (1992). Here thesingle stranded DNA is hybridized with a biotinylated oligonucleotidehaving a sequence corresponding to the 3′ end of the oligonucleotidetag. Preferably, the primer has a length of 20-25 bases. Clonesincluding a sequence complementary to the biotinylated oligonucleotideare selected by incubation with streptavidin coated magnetic beadsfollowed by magnetic capture. After capture of the positive clones, theplasmid DNA is released from the magnetic beads and converted intodouble stranded DNA using a DNA polymerase such as ThermoSequenase™(obtained from Amersham Pharmacia Biotech). Alternatively, protocolssuch as the Gene Trapper™ kit (Gibco BRL) can be used. The doublestranded DNA is then transformed, preferably by electroporation, intobacteria. The percentage of positive clones having the 5′ tagoligonucleotide is typically estimated to be between 90 and 98% from dotblot analysis.

Following transformation, the libraries are ordered in microtiter platesand sequenced. The Arabidopsis library was deposited at the AmericanType Culture Collection on Jan. 7, 2000 as “E-coli liba 010600” underthe accession number PTA-1161.

I. Example 2 Southern Hybridizations

The SDFs of the invention can be used in Southern hybridizations asdescribed above. The following describes extraction of DNA from nucleiof plant cells, digestion of the nuclear DNA and separation by length,transfer of the separated fragments to membranes, preparation of probesfor hybridization, hybridization and detection of the hybridized probe.

The procedures described herein can be used to isolate relatedpolynucleotides or for diagnostic purposes. Moderate stringencyhybridization conditions, as defined above, are described in the presentexample. These conditions result in detection of hybridization betweensequences having at least 70% sequence identity. As described above, thehybridization and wash conditions can be changed to reflect the desiredpercentage of sequence identity between probe and target sequences thatcan be detected.

In the following procedure, a probe for hybridization is produced fromtwo PCR reactions using two primers from genomic sequence of Arabidopsisthaliana. As described above, the particular template for generating theprobe can be any desired template.

The first PCR product is assessed to validate the size of the primer toassure it is of the expected size. Then the product of the first PCR isused as a template, with the same pair of primers used in the first PCR,in a second PCR that produces a labeled product used as the probe.

Fragments detected by hybridization, or other bands of interest, can beisolated from gels used to separate genomic DNA fragments by knownmethods for further purification and/or characterization.

Buffers for Nuclear DNA Extraction 1. 10×HB

1000 ml 40 mM 10.2 g Spermine (Sigma S-2876) and spermidine spermidine(Sigma S-2501) 10 mM spermine  3.5 g Stabilize chromatin and the nuclearmembrane 0.1M EDTA 37.2 g EDTA inhibits nuclease (disodium) 0.1M Tris12.1 g Buffer 0.8M KCl 59.6 g Adjusts ionic strength for stability ofnuclei

-   -   Adjust pH to 9.5 with 10 N NaOH. It appears that there is a        nuclease present in leaves. Use of pH 9.5 appears to inactivate        this nuclease.        2. 2 M sucrose (684 g per 1000 ml)    -   Heat about half the final volume of water to about 5° C. Add the        sucrose slowly then bring the mixture to close to final volume;        stir constantly until it has dissolved. Bring the solution to        volume.        3. Sarkosyl solution (lyses nuclear membranes)

1000 ml N-lauroyl sarcosine (Sarkosyl) 20.0 g 0.1M Tris 12.1 g 0.04MEDTA (Disodium) 14.9 g

-   -   Adjust the pH to 9.5 after all the components are dissolved and        bring up to the proper volume.        4.    -   20% Triton X-100    -   80 ml Triton X-100    -   320 ml 1×HB (w/o β-ME and PMSF)    -   Prepare in advance; Triton takes some time to dissolve

A. Procedure

-   1. Prepare 1X “H” buffer (keep ice-cold during use)

1000 ml 10X HB 100 ml 2 M sucrose 250 ml a non-ionic osmoticum Water 634ml Added just before use: 100 mM PMSF* 10 ml a protease inhibitor;protects nuclear membrane proteins β-mercaptoethanol 1 ml inactivatesnuclease by reducing disulfide bonds *100 mM PMSF (phenyl methylsulfonyl fluoride, Sigma P-7626) (add 0.0875 g to 5 ml 100% ethanol)

-   2. Homogenize the tissue in a blender (use 300-400 ml of 1×HB per    blender). Be sure that you use 5-10 ml of HB buffer per gram of    tissue. Blenders generate heat so be sure to keep the homogenate    cold. It is necessary to put the blenders in ice periodically.-   3. Add the 20% Triton X-100 (25 ml per liter of homogenate) and    gently stir on ice for 20 min. This lyses plastid, but not nuclear,    membranes.-   4. Filter the tissue suspension through several nylon filters into    an ice-cold beaker. The first filtration is through a 250-micron    membrane; the second is through an 85-micron membrane; the third is    through a 50-micron membrane; and the fourth is through a 20-micron    membrane. Use a large funnel to hold the filters. Filtration can be    sped up by gently squeezing the liquid through the filters.-   5. Centrifuge the filtrate at 1200×g for 20 min. at 4° C. to pellet    the nuclei.-   6. Discard the dark green supernatant. The pellet will have several    layers to it. One is starch; it is white and gritty. The nuclei are    gray and soft. In the early steps, there may be a dark green and    somewhat viscous layer of chloroplasts.    -   Wash the pellets in about 25 ml cold H buffer (with Triton        X-100) and resuspend by swirling gently and pipetting. After the        pellets are resuspended.    -   Pellet the nuclei again at 1200-1300×g. Discard the supernatant.    -   Repeat the wash 3-4 times until the supernatant has changed from        a dark green to a pale green. This usually happens after 3 or 4        resuspensions. At this point, the pellet is typically grayish        white and very slippery. The Triton X-100 in these repeated        steps helps to destroy the chloroplasts and mitochondria that        contaminate the prep.    -   Resuspend the nuclei for a final time in a total of 15 ml of H        buffer and transfer the suspension to a sterile 125 ml        Erlenmeyer flask.-   7. Add 15 ml, dropwise, cold 2% Sarkosyl, 0.1 M Tris, 0.04 M EDTA    solution (pH 9.5) while swirling gently. This lyses the nuclei. The    solution will become very viscous.-   8. Add 30 grams of CsCl and gently swirl at room temperature until    the CsCl is in solution. The mixture will be gray, white and    viscous.-   9. Centrifuge the solution at 11,400×g at 4° C. for at least 30 min.    The longer this spin is, the firmer the protein pellicle.-   10. The result is typically a clear green supernatant over a white    pellet, and (perhaps) under a protein pellicle. Carefully remove the    solution under the protein pellicle and above the pellet. Determine    the density of the solution by weighing 1 ml of solution and add    CsCl if necessary to bring to 1.57 g/ml. The solution contains    dissolved solids (sucrose etc) and the refractive index alone will    not be an accurate guide to CsCl concentration.-   11. Add 20 μl of 10 mg/ml EtBr per ml of solution.-   12. Centrifuge at 184,000×g for 16 to 20 hours in a fixed-angle    rotor.-   13. Remove the dark red supernatant that is at the top of the tube    with a plastic transfer pipette and discard. Carefully remove the    DNA band with another transfer pipette. The DNA band is usually    visible in room light; otherwise, use a long wave UV light to locate    the band.-   14. Extract the ethidium bromide with isopropanol saturated with    water and salt. Once the solution is clear, extract at least two    more times to ensure that all of the EtBr is gone. Be very gentle,    as it is very easy to shear the DNA at this step. This extraction    may take a while because the DNA solution tends to be very viscous.    If the solution is too viscous, dilute it with TE.-   15. Dialyze the DNA for at least two days against several changes    (at least three times) of TE (10 mM Tris, 1 mM EDTA, pH 8) to remove    the cesium chloride.-   16. Remove the dialyzed DNA from the tubing. If the dialyzed DNA    solution contains a lot of debris, centrifuge the DNA solution at    least at 2500×g for 10 min. and carefully transfer the clear    supernatant to a new tube. Read the A260 concentration of the DNA.-   17. Assess the quality of the DNA by agarose gel electrophoresis (1%    agarose gel) of the DNA. Load 50 ng and 100 ng (based on the OD    reading) and compare it with known and good quality DNA. Undigested    lambda DNA and a lambda-HindIII-digested DNA are good molecular    weight makers.

Protocol for Digestion of Genomic DNA Protocol:

-   1. The relative amounts of DNA for different crop plants that    provide approximately a balanced number of genome equivalent is    given in Table 3. Note that due to the size of the wheat genome,    wheat DNA will be underrepresented. Lambda DNA provides a useful    control for complete digestion.-   2. Precipitate the DNA by adding 3 volumes of 100% ethanol. Incubate    at −20° C. for at least two hours. Yeast DNA can be purchased and    made up at the necessary concentration, therefore no precipitation    is necessary for yeast DNA.-   3. Centrifuge the solution at 11,400×g for 20 min. Decant the    ethanol carefully (be careful not to disturb the pellet). Be sure    that the residual ethanol is completely removed either by vacuum    desiccation or by carefully wiping the sides of the tubes with a    clean tissue.-   4. Resuspend the pellet in an appropriate volume of water. Be sure    the pellet is fully resuspended before proceeding to the next step.    This may take about 30 min.-   5. Add the appropriate volume of 10× reaction buffer provided by the    manufacturer of the restriction enzyme to the resuspended DNA    followed by the appropriate volume of enzymes. Be sure to mix it    properly by slowly swirling the tubes.-   6. Set-up the lambda digestion-control for each DNA that you are    digesting.-   7. Incubate both the experimental and lambda digests overnight at    37° C. Spin down condensation in a microfuge before proceeding.-   8. After digestion, add 2 μl of loading dye (typically 0.25%    bromophenol blue, 0.25% xylene cyanol in 15% Ficoll or 30% glycerol)    to the lambda-control digests and load in 1% TPE-agarose gel (TPE is    90 mM Tris-phosphate, 2 mM EDTA, pH 8). If the lambda DNA in the    lambda control digests are completely digested, proceed with the    precipitation of the genomic DNA in the digests.-   9. Precipitate the digested DNA by adding 3 volumes of 100% ethanol    and incubating in −20° C. for at least 2 hours (preferably    overnight).    -   EXCEPTION: Arabidopsis and yeast DNA are digested in an        appropriate volume; they don't have to be precipitated.-   10. Resuspend the DNA in an appropriate volume of TE (e.g., 22 μl×50    blots=1100 μl) and an appropriate volume of 10× loading dye (e.g.,    2.4 μl×50 blots=120 μl). Be careful in pipetting the loading dye—it    is viscous. Be sure you are pipetting the correct volume.

TABLE 3 Some guide points in digesting genomic DNA. Genome SizeEquivalent Amount Relative to to 2 μg of DNA Species Genome SizeArabidopsis Arabidopsis DNA per blot Arabidopsis 120 Mb 1X   1X    2 μgBrassica 1,100 Mb 9.2X 0.54X 10 μg Corn 2,800 Mb 23.3X  0.43X 20 μgCotton 2,300 Mb 19.2X  0.52X 20 μg Oat 11,300 Mb 94X   0.11X 20 μg Rice400 Mb 3.3X 0.75X  5 μg Soybean 1,100 Mb 9.2X 0.54X 10 μg Sugarbeet 758Mb 6.3X 0.8X  10 μg Sweetclover 1,100 Mb 9.2X 0.54X 10 μg Wheat 16,000Mb 133X    0.08X 20 μg Yeast 15 Mb  0.12X 1X   0.25 μg  

Protocol for Southern Blot Analysis

The digested DNA samples are electrophoresed in 1% agarose gels in 1×TPEbuffer. Low voltage; overnight separations are preferred. The gels arestained with EtBr and photographed.

-   1. For blotting the gels, first incubate the gel in 0.25 N HCl (with    gentle shaking) for about 15 min.-   2. Then briefly rinse with water. The DNA is denatured by 2    incubations. Incubate (with shaking) in 0.5 M NaOH in 1.5 M NaCl for    15 min.-   3. The gel is then briefly rinsed in water and neutralized by    incubating twice (with shaking) in 1.5 M Tris pH 7.5 in 1.5 M NaCl    for 15 min.-   4. A nylon membrane is prepared by soaking it in water for at least    5 min, then in 6×SSC for at least 15 min. before use. (20×SSC is    175.3 g NaCl, 88.2 g sodium citrate per liter, adjusted to pH 7.0.)-   5. The nylon membrane is placed on top of the gel and all bubbles in    between are removed. The DNA is blotted from the gel to the membrane    using an absorbent medium, such as paper toweling and 6×SCC buffer.    After the transfer, the membrane may be lightly brushed with a    gloved hand to remove any agarose sticking to the surface.-   6. The DNA is then fixed to the membrane by UV crosslinking and    baking at 80° C. The membrane is stored at 4° C. until use.

B. Protocol for PCR Amplification of Genomic Fragments in Arabidopsis

Amplification Procedures:

1. Mix the following in a 0.20 ml PCR tube or 96-well PCR plate:

Volume Stock Final Amount or Conc. 0.5 μl ~10 ng/μl genomic DNA¹ 5 ng2.5 μl 10× PCR buffer 20 mM Tris, 50 mM KCl 0.75 μl 50 mM MgCl₂ 1.5 mM 1μl 10 pmol/μl Primer 1 (Forward) 10 pmol 1 μl 10 pmol/μl Primer 2(Reverse) 10 pmol 0.5 μl 5 mM dNTPs 0.1 mM 0.1 μl 5 units/μl PlatinumTaq ™ (Life 1 units Technologies, Gaithersburg, MD) DNA Polymerase (to25 μl) Water ¹ Arabidopsis DNA is used in the present experiment, butthe procedure is a general one.

2. The template DNA is amplified using a Perkin Elmer 9700 PCR machine:

1) 94° C. for 10 min. followed by

2) 3) 4) 5 cycles: 5 cycles: 25 cycles: 94° C. - 30 sec 94° C. - 30 sec94° C. - 30 sec 62° C. - 30 sec 58° C. - 30 sec 53° C. - 30 sec 72° C. -3 min 72° C. - 3 min 72° C. - 3 min

5) 72° C. for 7 min. Then the reactions are stopped by chilling to 4° C.

The procedure can be adapted to a multi-well format if necessary.

Quantification and Dilution of PCR Products:

-   1. The product of the PCR is analyzed by electrophoresis in a 1%    agarose gel. A linearized plasmid DNA can be used as a    quantification standard (usually at 50, 100, 200, and 400 ng). These    will be used as references to approximate the amount of PCR    products. HindIII-digested Lambda DNA is useful as a molecular    weight marker. The gel can be run fairly quickly; e.g., at 100    volts. The standard gel is examined to determine that the size of    the PCR products is consistent with the expected size and if there    are significant extra bands or smeary products in the PCR reactions.-   2. The amounts of PCR products can be estimated on the basis of the    plasmid standard.-   3. For the small number of reactions that produce extraneous bands,    a small amount of DNA from bands with the correct size can be    isolated by dipping a sterile 10-μl tip into the band while viewing    though a UV Transilluminator. The small amount of agarose gel (with    the DNA fragment) is used in the labeling reaction.

C. Protocol for PCR-DIG-Labeling of DNA Solutions:

-   -   Reagents in PCR reactions (diluted PCR products, 10×PCR Buffer,        50 mM MgCl₂, 5 U/μl Platinum Taq Polymerase, and the primers)    -   10× dNTP+DIG-11-dUTP [1:5]: (2 mM dATP, 2 mM dCTP, 2 mM dGTP,        1.65 mM dTTP, 0.35 mM DIG-11-dUTP)    -   10× dNTP+DIG-11-dUTP [1:10]: (2 mM dATP, 2 mM dCTP, 2 mM dGTP,        1.81 mM dTTP, 0.19 mM DIG-11-dUTP)    -   10× dNTP+DIG-11-dUTP [1:15]: (2 mM dATP, 2 mM dCTP, 2 mM dGTP,        1.875 mM dTTP, 0.125 mM DIG-11-dUTP)    -   TE buffer (10 mM Tris, 1 mM EDTA, pH 8)    -   Maleate buffer: In 700 ml of deionized distilled water, dissolve        11.61 g maleic acid and 8.77 g NaCl. Add NaOH to adjust the pH        to 7.5. Bring the volume to 1 L. Stir for 15 min. and sterilize.    -   10% blocking solution: In 80 ml deionized distilled water,        dissolve 1.16 g maleic acid. Next, add NaOH to adjust the pH to        7.5. Add 10 g of the blocking reagent powder (Boehringer        Mannheim, Indianapolis, Ind., Cat. no. 1096176). Heat to 60° C.        while stirring to dissolve the powder. Adjust the volume to 100        ml with water. Stir and sterilize.    -   1% blocking solution: Dilute the 10% stock to 1% using the        maleate buffer.    -   Buffer 3 (100 mM Tris, 100 mM NaCl, 50 mM MgCl₂, pH9.5).        Prepared from autoclaved solutions of 1M Tris pH 9.5, 5 M NaCl,        and 1 M MgCl₂ in autoclaved distilled water.

Procedure:

-   1. PCR reactions are performed in 25 μl volumes containing:

PCR buffer 1X MgCl₂ 1.5 mM 10X dNTP + DIG-11-dUTP 1X (please see thenote below) Platinum Taq ™ Polymerase 1 unit 10 pg probe DNA 10 pmolprimer 1 Note: Use for: 10X dNTP + DIG-11-dUTP (1:5) <1 kb 10X dNTP +DIG-11-dUTP (1:10)  1 kb to 1.8 kb 10X dNTP + DIG-11-dUTP (1:15) >1.8 kb

-   2. The PCR reaction uses the following amplification cycles:    -   1) 94° C. for 10 min.

2) 3) 4) 5 cycles: 5 cycles: 25 cycles: 95° C. - 30 sec 95° C. - 30 sec95° C. - 30 sec 61° C. - 1 min 59° C. - 1 min 51° C. - 1 min 73° C. - 5min 75° C. - 5 min 73° C. - 5 min

-   -   5) 72° C. for 8 min. The reactions are terminated by chilling to        4° C. (hold).

-   3. The products are analyzed by electrophoresis—in a 1% agarose gel,    comparing to an aliquot of the unlabelled probe starting material.

-   4. The amount of DIG-labeled probe is determined as follows:    -   Make serial dilutions of the diluted control DNA in dilution        buffer (TE: 10 mM Tris and 1 mM EDTA, pH 8) as shown in the        following table:

DIG-labeled control Final Conc. (Dilution DNA starting conc. StepwiseDilution Name)  5 ng/μl  1 μl in 49 μl TE 100 pg/μl (A) 100 pg/μl (A) 25μl in 25 μl TE  50 pg/μl (B)  50 pg/μl (B) 25 μl in 25 μl TE  25 pg/μl(C)  25 pg/μl (C) 20 μl in 30 μl TE  10 pg/μl (D)

-   -   a. Serial deletions of a DIG-labeled standard DNA ranging from        100 pg to 10 pg are spotted onto a positively charged nylon        membrane, marking the membrane lightly with a pencil to identify        each dilution.    -   b. Serial dilutions (e.g., 1:50, 1:2500, 1:10,000) of the newly        labeled DNA probe are spotted.    -   c. The membrane is fixed by UV crosslinking.    -   d. The membrane is wetted with a small amount of maleate buffer        and then incubated in 1% blocking solution for 15 min at room        temp.    -   e. The labeled DNA is then detected using alkaline phosphatase        conjugated anti-DIG antibody (Boehringer Mannheim, Indianapolis,        Ind., cat. no. 1093274) and an NBT substrate according to the        manufacture's instruction.    -   f. Spot intensities of the control and experimental dilutions        are then compared to estimate the concentration of the        PCR-DIG-labeled probe.

D. Prehybridization and Hybridization of Southern Blots Solutions:

100% Formamide purchased from Gibco 20X SSC (1X = 0.15M NaCl, 0.015MNa₃citrate) per L: 175 g NaCl 87.5 g Na₃citrate 2H₂0

-   -   20% Sarkosyl (N-lauroyl-sarcosine)    -   20% SDS (sodium dodecyl sulphate)    -   10% Blocking Reagent: In 80 ml deionized distilled water,        dissolve 1.16 g maleic acid. Next, add NaOH to adjust the pH to        7.5. Add 10g of the blocking reagent powder. Heat to 60° C.        while stirring to dissolve the powder. Adjust the volume to 100        ml with water. Stir and sterilize.

Prehybridization Mix:

Final Volume Concentration Components (per 100 ml) Stock   50% Formamide 50 ml 100%  5X SSC  25 ml 20X  0.1% Sarkosyl 0.5 ml 20% 0.02% SDS 0.1ml 20%   2% Blocking Reagent  20 ml 10% Water 4.4 ml

General Procedures:

-   1. Place the blot in a heat-sealable plastic bag and add an    appropriate volume of prehybridization solution (30 ml/100 cm²) at    room temperature. Seal the bag with a heat sealer, avoiding bubbles    as much as possible. Lay down the bags in a large plastic tray (one    tray can accommodate at least 4-5 bags). Ensure that the bags are    lying flat in the tray so that the prehybridization solution is    evenly distributed throughout the bag. Incubate the blot for at    least 2 hours with gentle agitation using a waver shaker.-   2. Denature DIG-labeled DNA probe by incubating for 10 min. at    98° C. using the PCR machine and immediately cool it to 4° C.-   3. Add probe to prehybridization solution (25 ng/ml; 30 ml=750 ng    total probe) and mix well but avoid foaming. Bubbles may lead to    background.-   4. Pour off the prehybridization solution from the hybridization    bags and add new prehybridization and probe solution mixture to the    bags containing the membrane.-   5. Incubate with gentle agitation for at least 16 hours.-   6. Proceed to medium stringency post-hybridization wash:    -   Three times for 20 min. each with gentle agitation using 1×SSC,        1% SDS at 60° C.    -   All wash solutions must be prewarmed to 60° C. Use about 100 ml        of wash solution per membrane.    -   To avoid background keep the membranes fully submerged to avoid        drying in spots; agitate sufficiently to avoid having membranes        stick to one another.-   7. After the wash, proceed to immunological detection and CSPD    development.    E. Procedure for Immunological Detection with CSPD

Solutions:

Buffer 1: Maleic acid buffer (0.1M maleic acid, 0.15M NaCl; adjusted topH 7.5 with NaoH) Washing buffer: Maleic acid buffer with 0.3% (v/v)Tween 20. Blocking stock 10% blocking reagent in buffer 1. Dissolve (10Xsolution concentration): blocking reagent powder (Boehringer Mannheim,Indianapolis, IN, cat. no. 1096176) by constantly stirring on a 65° C.heating block or heat in a microwave, autoclave and store at 4° C.Buffer 2 Dilute the stock solution 1:10 in Buffer 1. (1X blockingsolution): Detection buffer: 0.1M Tris, 0.1M NaCl, pH 9.5

Procedure:

-   1. After the post-hybridization wash the blots are briefly rinsed    (1-5 min.) in the maleate washing buffer with gentle shaking.-   2. Then the membranes are incubated for 30 min. in Buffer 2 with    gentle shaking.-   3. Anti-DIG-AP conjugate (Boehringer Mannheim, Indianapolis, Ind.,    cat. no. 1093274) at 75 mU/ml (1:10,000) in Buffer 2 is used for    detection. 75 ml of solution can be used for 3 blots.-   4. The membrane is incubated for 30 min. in the antibody solution    with gentle shaking.-   5. The membrane are washed twice in washing buffer with gentle    shaking. About 250 mls is used per wash for 3 blots.-   6. The blots are equilibrated for 2-5 min in 60 ml detection buffer.-   7. Dilute CSPD (1:200) in detection buffer. (This can be prepared    ahead of time and stored in the dark at 4° C.).    -   The following steps must be done individually. Bags (one for        detection and one for exposure) are generally cut and ready        before doing the following steps.-   8. The blot is carefully removed from the detection buffer and    excess liquid removed without drying the membrane. The blot is    immediately placed in a bag and 1.5 ml of CSPD solution is added.    The CSPD solution can be spread over the membrane. Bubbles present    at the edge and on the surface of the blot are typically removed by    gentle rubbing. The membrane is incubated for 5 min. in CSPD    solution.-   9. Excess liquid is removed and the membrane is blotted briefly (DNA    side up) on Whatman 3MM paper. Do not let the membrane dry    completely.-   10. Seal the damp membrane in a hybridization bag and incubate for    10 min at 37° C. to enhance the luminescent reaction.-   11. Expose for 2 hours at room temperature to X-ray film. Multiple    exposures can be taken. Luminescence continues for at least 24 hours    and signal intensity increases during the first hours.

Example 3 Microarray Experiments and Results Example 3 MicroarrayExperiments and Results 1. Sample Tissue Preparation (a) Roots

Seeds of Arabidopsis thaliana (Ws) were sterilized in full strengthbleach for less than 5 min., washed more than 3 times in steriledistilled deionized water and plated on MS agar plates. The plates wereplaced at 4° C. for 3 nights and then placed vertically into a growthchamber having 16 hr light/8 hr dark cycles, 23° C., 70% relativehumidity and ˜11,000 LUX. After 2 weeks, the roots were cut from theagar, flash frozen in liquid nitrogen and stored at −80° C. (EXPT REP:108439 and 108434)

(b) Root Hairless Mutants

Plants mutant at the rhl gene locus lack root hairs. This mutation ismaintained as a heterozygote.

Seeds of Arabidopsis thaliana (Landsberg erecta) mutated at the rhi genelocus were sterilized using 30% bleach with 1 ul/ml 20% Triton-X 100 andthen vernalized at 4° C. for 3 days before being plated onto GM agarplates. Plates were placed in growth chamber with 16 hr light/8 hr.dark, 23° C., 14,500-15,900 LUX, and 70% relative humidity forgermination and growth.

After 7 days, seedlings were inspected for root hairs using a dissectingmicroscope. Mutants were harvested and the cotyledons removed so thatonly root tissue remained. Tissue was then flash frozen in liquidnitrogen and stored at −80 C. (EXPT REP: 108433) Arabidopsis thaliana(Landsberg erecta) seedlings grown and prepared as above were used ascontrols. (EXPT REP: 108433)

Alternatively, seeds of Arabidopsis thaliana (Landsberg erecta),heterozygous for the rhl1 (root hairless) mutation, weresurface-sterilized in 30% bleach containing 0.1% Triton X-100 andfurther rinsed in sterile water. They were then vernalized at 4° C. for4 days before being plated onto MS agar plates. The plates weremaintained in a growth chamber at 24° C. with 16 hr light/8 hr dark forgermination and growth. After 10 days, seedling roots that expressed thephenotype (i.e. lacking root hairs) were cut below the hypocotyljunction, frozen in liquid nitrogen and stored at −80° C. Thoseseedlings with the normal root phenotype (heterozygous or wt) werecollected as described for the mutant and used as controls.

(c) Rosette Leaves, Stems, and Siliques

Arabidopsis thaliana (Ws) seed was vernalized at 4° C. for 3 days beforesowing in Metro-mix soil type 350. Flats were placed in a growth chamberhaving 16 hr light/8 hr dark, 80% relative humidity, 23° C. and 13,000LUX for germination and growth. After 3 weeks, rosette leaves, stems,and siliques (see EXPT REP: 108436, 108437 and 108438) were harvested,flash frozen in liquid nitrogen and stored at −80° C. until use. After 4weeks, siliques (<5 mm, 5-10 mm and >10 mm) were harvested, flash frozenin liquid nitrogen and stored at −80° C. until use. 5 week old wholeplants (used as controls) were harvested, flash frozen in liquidnitrogen and kept at −80° C. until RNA was isolated.

(d) Trichomes

Arabidopsis thaliana (Colombia glabrous) inflorescences were used as acontrol and CS8143 (hairy inflorescence ecotype) inflorescences, havingincreased trichomes, were used as the experimental sample.

Approximately 10 μl of each type of seed was sown on a flat of 350 soil(containing 0.03% marathon) and vernalized at 4° C. for 3 days. Plantswere then grown at room temperature under florescent lighting. Younginflorescences were collected at 30 days for the control plants and 37days for the experimental plants. Each inflorescence was cut intoone-half inch (½″) pieces, flash frozen in liquid nitrogen and stored at−80° C. until RNA was isolated.

(e) Germination

Arabidopsis thaliana seeds (ecotype Ws) were sterilized in bleach andrinsed with sterile water. The seeds were placed in 100 mm petri platescontaining soaked autoclaved filter paper. Plates were foil-wrapped andleft at 4° C. for 3 nights to vernalize. After cold treatment, the foilwas removed and plates were placed into a growth chamber having 16 hrlight/8 hr dark cycles, 23° C., 70% relative humidity and ˜11,000 lux.Seeds were collected 1 d (EXPT REP: 108461), 2 d (EXPT REP: 108462), 3 d(EXPT REP: 108463) and 4 d (EXPT REP: 108464) later, flash frozen inliquid nitrogen and stored at −80° C. until RNA was isolated.

(f) Shoot Apical Meristem

Arabidopsis thaliana (Landsberg erecta) plants mutant at the stm genelocus lack shoot meristems, produce aerial rosettes, have a reducednumber of flowers per inflorescence, as well as a reduced number ofpetals, stamens and carpels, and is female sterile. This mutation ismaintained as a heterozygote.

Seeds of Arabidopsis thaliana (Landsberg erecta) mutated at the stmlocus were sterilized using 30% bleach with 1 ul/ml 20% Triton-X100. Theseeds were vernalized at 4° C. for 3 days before being plated onto GMagar plates. Half were then put into a 22° C., 24 hr light growthchamber and half in a 24° C. 16 hr light/8 hr dark growth chamber having14,500-15,900 LUX, and 70% relative humidity for germination and growth.

After 7 days, seedlings were examined for leaf primordia using adissecting microscope. Presence of leaf primordia indicated a wild typephenotype. Mutants were selected based on lack of leaf primordia.Mutants were then harvested and hypocotyls removed leaving only tissuein the shoot region. Tissue was then flash frozen in liquid nitrogen andstored at −80° C.

Control tissue was isolated from 5 day old Landsberg erecta seedlingsgrown in the same manner as above. Tissue from the shoot region washarvested in the same manner as the stm tissue, but only containedmaterial from the 24° C., 16 hr light/8 hr dark long day cycle growthchamber. (EXPT REP: 108453)

Seeds of maize hybrid 35A (Pioneer) were sown in water-moistened sand inflats (10 rows, 5-6 seed/row) and covered with clear, plastic lidsbefore being placed in a growth chamber having 16 hr light (25° C.)/8 hrdark (20° C.), 75% relative humidity and 13,000-14,000 LUX. Coveredflats were watered every three days for 8 days. Seedlings were carefullyremoved from the sand and the outer layers of leaf shealth removed.About 2 mm sections were cut and flash frozen in liquid nitrogen priorto storage at −80° C. The tissues above the shoot apices (˜1 cm long)were cut, treated as above and used as control tissue.

(g) Abscissic Acid (ABA)

Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were sown in traysand left at 4° C. for 4 days to vernalize. They were then transferred toa growth chamber having grown 16 hr light/8 hr dark, 13,000 LUX, 70%humidity, and 20° C. and watered twice a week with 1 L of 1× Hoagland'ssolution. Approximately 1,000 14 day old plants were spayed with 200-250mls of 100 μM ABA in a 0.02% solution of the detergent Silwet L-77.Whole seedlings, including roots, were harvested within a 15 to 20minute time period at 1 hr and 6 hr after treatment, flash-frozen inliquid nitrogen and stored at −80° C.

Seeds of maize hybrid 35A (Pioneer) were sown in water-moistened sand inflats (10 rows, 5-6 seed/row) and covered with clear, plastic lidsbefore being placed in a growth chamber having 16 hr light (25° C.)/8 hrdark (20° C.), 75% relative humidity and 13,000-14,000 LUX. Coveredflats were watered every three days for 7 days. Seedlings were carefullyremoved from the sand and placed in 1-liter beakers with 100 μM ABA fortreatment. Control plants were treated with water. After 6 hr and 24 hr,aerial and root tissues were separated and flash frozen in liquidnitrogen prior to storage at −80° C.

(h) Auxin Responsive

Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were sown in traysand left at 4° C. for 4 days to vernalize. They were then transferred toa growth chamber having 16 hr light/8 hr dark, 13,000 LUX, 70% humidity,20° C. and watered twice a week with 1 L of 1× Hoagland's solution(recipe recited in Feldmann et al., (1987) Mol. Gen. Genet. 208: 1-9 anddescribed as complete nutrient solution). Approximately 1,000 14 day oldplants were spayed with 200-250 mls of 100 μM NAA in a 0.02% solution ofthe detergent Silwet L-77. Aerial tissues (everything above the soilline) were harvested within a 15 to 20 minute time period 1 hr and 6 hrsafter treatment, flash-frozen in liquid nitrogen and stored at −80° C.

Seeds of maize hybrid 35A (Pioneer) were sown in water-moistened sand inflats (10 rows, 5-6 seed/row) and covered with clear, plastic lidsbefore being placed in a growth chamber having 16 hr light (25° C.)/8 hrdark (20° C.), 75% relative humidity and 13,000-14,000 LUX. Coveredflats were watered every three days for 7 days. Seedlings were carefullyremoved from the sand and placed in 1-liter beakers with 100 μM NAA fortreatment. Control plants were treated with water. After 6 hr and 24 hr,aerial and root tissues were separated and flash frozen in liquidnitrogen prior to storage at −80° C.

(i) Cytokinin

Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were sown in traysand left at 4° C. for 4 days to vernalize. They were then transferred toa growth chamber having 16 hr light/8 hr dark, 13,000 LUX, 70% humidity,20° C. temperature and watered twice a week with 1 L of 1× Hoagland'ssolution. Approximately 1,000 14 day old plants were spayed with 200-250mls of 100 μM BA in a 0.02% solution of the detergent Silwet L-77.Aerial tissues (everything above the soil line) were harvested within a15 to 20 minute time period 1 hr and 6 hrs after treatment, flash-frozenin liquid nitrogen and stored at −80° C.

Seeds of maize hybrid 35A (Pioneer) were sown in water-moistened sand inflats (10 rows, 5-6 seed/row) and covered with clear, plastic lidsbefore being placed in a growth chamber having 16 hr light (25° C.)/8 hrdark (20° C.), 75% relative humidity and 13,000-14,000 LUX. Coveredflats were watered every three days for 7 days. Seedlings were carefullyremoved from the sand and placed in 1-liter beakers with 100 μM BA fortreatment. Control plants were treated with water. After 6 hr, aerialand root tissues were separated and flash frozen in liquid nitrogenprior to storage at −80° C.

(j) Brassinosteroid Responsive

Two separate experiments were performed, one with epi-brassinolide andone with the brassinosteroid biosynthetic inhibitor brassinazole.

In the epi-brassinolide experiments, seeds of wild-type Arabidopsisthaliana (ecotype Wassilewskija) and the brassinosteroid biosyntheticmutant dwf4-1 were sown in trays and left at 4° C. for 4 days tovernalize. They were then transferred to a growth chamber having 16 hrlight/8 hr dark, 11,000 LUX, 70% humidity and 22° C. temperature. Fourweek old plants were spayed with a 1 μM solution of epi-brassinolide andshoot parts (unopened floral primordia and shoot apical meristems)harvested three hours later. Tissue was flash-frozen in liquid nitrogenand stored at −80° C. (EXPT REP 108480)

In the brassinazole experiments, seeds of wild-type Arabidopsis thaliana(ecotype Wassilewskija) were grown as described above. Four week oldplants were spayed with a 1 μM solution of brassinazole and shoot parts(unopened floral primordia and shoot apical meristems) harvested threehours later. Tissue was flash-frozen in liquid nitrogen and stored at−80° C. (EXPT REP 108481)

In addition to the spray experiments, tissue was prepared from twodifferent mutants; (1) a dwf4-1 knock out mutant (EXPT REP: 108478) and(2) a mutant overexpressing the dwf4-1 gene (EXPT REP: 108479).

Seeds of wild-type Arabidopsis thaliana (ecotype Wassilewskija) and ofthe dwf4-1 knock out and overexpressor mutants were sown in trays andleft at 4° C. for 4 days to vernalize. They were then transferred to agrowth chamber having 16 hr light/8 hr dark, 11,000 LUX, 70% humidityand 22° C. temperature. Tissue from shoot parts (unopened floralprimordia and shoot apical meristems) was flash-frozen in liquidnitrogen and stored at −80° C.

Another experiment was completed with seeds of Arabidopsis thaliana(ecotype Wassilewskija) were sown in trays and left at 4° C. for 4 daysto vernalize. They were then transferred to a growth chamber. Plantswere grown under long-day (16 hr light: 8 hr. dark) conditions, 13,000LUX light intensity, 70% humidity, 20° C. temperature and watered twicea week with 1 L 1× Hoagland's solution (recipe recited in Feldmann etal., (1987) Mol. Gen. Genet. 208: 1-9 and described as complete nutrientsolution). Approximately 1,000 14 day old plants were spayed with200-250 mls of 0.1 μM Epi-Brassinolite in 0.02% solution of thedetergent Silwet L-77. At 1 hr. and 6 hrs. after treatment aerialtissues were harvested within a 15 to 20 minute time period andflash-frozen in liquid nitrogen.

Seeds of maize hybrid 35A (Pioneer) were sown in water-moistened sand inflats (10 rows, 5-6 seed/row) and covered with clear, plastic lidsbefore being placed in a growth chamber having 16 hr light (25° C.)/8 hrdark (20° C.), 75% relative humidity and 13,000-14,000 LUX. Coveredflats were watered every three days for 7 days. Seedlings were carefullyremoved from the sand and placed in 1-liter beakers with 0.1 μMepi-brassinolide for treatment. Control plants were treated withdistilled deionized water. After 24 hr, aerial and root tissues wereseparated and flash frozen in liquid nitrogen prior to storage at −80°C.

(k) Gibberillic Acid

Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were sown in traysand left at 4° C. for 4 days to vernalize. They were then transferred toa growth chamber having 16 hr light/8 hr. dark, 13,000 LUX, 70%humidity, 20° C. and watered twice a week with 1 L of 1× Hoagland'ssolution. Approximately 1,000 14 day old plants were spayed with 200-250mls of 100 μM gibberillic acid in a 0.02% solution of the detergentSilwet L-77. At 1 hr. and 6 hrs. after treatment, aerial tissues(everything above the soil line) were harvested within a 15 to 20 minutetime period, flash-frozen in liquid nitrogen and stored at −80° C.

Alternatively, seeds of Arabidopsis thaliana (ecotype Ws) were sown inMetro-mix soil type 350 and left at 4° C. for 3 days to vernalize. Theywere then transferred to a growth chamber having 16 hr light/8 hr dark,13,000 LUX, 80% humidity, 20° C. temperature and watered every four dayswith 1.5 L water. 14 days after germination, plants were sprayed with100 μM gibberillic acid or with water. Aerial tissues were harvested 1hr (EXPT REP: 108484), 6 hrs (EXPT REP: 108485), 12 hrs (EXPT REP:108486), and 24 hrs post-treatment, flash frozen and stored at −80° C.

Seeds of maize hybrid 35A (Pioneer) were sown in water-moistened sand inflats (10 rows, 5-6 seed/row) and covered with clear, plastic lidsbefore being placed in a growth chamber having 16 hr light (25° C.)/8 hrdark (20° C.), 75% relative humidity and 13,000-14,000 LUX. Coveredflats were watered every three days for 7 days. Seedlings were carefullyremoved from the sand and placed in 1-liter beakers with 100 μMgibberillic acid for treatment. Control plants were treated with water.After 1 hr, 6 hr and 12 hr, aerial and root tissues were separated andflash frozen in liquid nitrogen prior to storage at −80° C.

(l) Nitrogen: High to Low

Wild type Arabidopsis thaliana seeds (ecotpye Ws) were surfacesterilized with 30% Clorox, 0.1% Triton X-100 for 5 minutes. Seeds werethen rinsed with 4-5 exchanges of sterile double distilled deionizedwater. Seeds were vernalized at 4° C. for 2-4 days in darkness. Aftercold treatment, seeds were plated on modified 1×MS media (without NH₄NO₃or KNO₃), 0.5% sucrose, 0.5 g/L MES pH5.7, 1% phytagar and supplementedwith KNO₃ to a final concentration of 60 mM (high nitrate modified 1×MSmedia). Plates were then grown for 7 days in a Percival growth chamberat 22° C. with 16 hr. light/8 hr dark.

Germinated seedlings were then transferred to a sterile flask containing50 mL of high nitrate modified 1×MS liquid media. Seedlings were grownwith mild shaking for 3 additional days at 22° C. in 16 hr. light/8 hrdark (in a Percival growth chamber) on the high nitrate modified 1×MSliquid media.

After three days of growth on high nitrate modified 1×MS liquid media,seedlings were transferred either to a new sterile flask containing 50mL of high nitrate modified 1×MS liquid media or to low nitrate modified1×MS liquid media (containing 20 □M KNO₃). Seedlings were grown in thesemedia conditions with mild shaking at 22° C. in 16 hr light/8 hr darkfor the appropriate time points and whole seedlings harvested for totalRNA isolation via the Trizol method (LifeTech). The time points used forthe microarray experiments were 10 min. (EXPT REP: 108454) and 1 hour(EXPT REP: 108455) time points for both the high and low nitratemodified 1×MS media.

Alternatively, seeds that were surface sterilized in 30% bleachcontaining 0.1% Triton X-100 and further rinsed in sterile water, wereplanted on MS agar, (0.5% sucrose) plates containing 50 mM KNO₃(potassium nitrate). The seedlings were grown under constant light (3500LUX) at 22° C. After 12 days, seedlings were transferred to MS agarplates containing either 1 mM KNO₃ or 50 mM KNO₃. Seedlings transferredto agar plates containing 50 mM KNO₃ were treated as controls in theexperiment. Seedlings transferred to plates with 1 mM KNO₃ were rinsedthoroughly with sterile MS solution containing 1 mM KNO₃. There were tenplates per transfer. Root tissue was collected and frozen in 15 mLFalcon tubes at various time points which included 1 hour, 2 hours, 3hours, 4 hours, 6 hours, 9 hours, 12 hours, 16 hours, and 24 hours.

Maize 35A19 Pioneer hybrid seeds were sown on flats containing sand andgrown in a Conviron growth chamber at 25° C., 16 hr light/8 hr dark,˜13,000 LUX and 80% relative humidity. Plants were watered every threedays with double distilled deionized water. Germinated seedlings areallowed to grow for 10 days and were watered with high nitrate modified1×MS liquid media (see above). On day 11, young corn seedlings wereremoved from the sand (with their roots intact) and rinsed briefly inhigh nitrate modified 1×MS liquid media. The equivalent of half a flatof seedlings were then submerged (up to their roots) in a beakercontaining either 500 mL of high or low nitrate modified 1×MS liquidmedia (see above for details).

At appropriate time points, seedlings were removed from their respectiveliquid media, the roots separated from the shoots and each tissue typeflash frozen in liquid nitrogen and stored at −80° C. This was repeatedfor each time point. Total RNA was isolated using the Trizol method (seeabove) with root tissues only.

Corn root tissues isolated at the 4 hr and 16 hr time points were usedfor the microarray experiments. Both the high and low nitrate modified1×MS media were used.

(m) Nitrogen: Low to High

Arabidopsis thaliana ecotype Ws seeds were sown on flats containing 4 Lof a 1:2 mixture of Grace Zonolite vermiculite and soil. Flats werewatered with 3 L of water and vernalized at 4° C. for five days. Flatswere placed in a Conviron growth chamber having 16 hr light/8 hr dark at20° C., 80% humidity and 17,450 LUX. Flats were watered withapproximately 1.5 L of water every four days. Mature, bolting plants (24days after germination) were bottom treated with 2 L of either a control(100 mM mannitol pH 5.5) or an experimental (50 mM ammonium nitrate, pH5.5) solution. Roots, leaves and siliques were harvested separately 30,120 and 240 minutes after treatment, flash frozen in liquid nitrogen andstored at −80° C.

Hybrid maize seed (Pioneer hybrid 35A19) were aerated overnight indeionized water. Thirty seeds were plated in each flat, which contained4 liters of Grace zonolite vermiculite. Two liters of water were bottomfed and flats were kept in a Conviron growth chamber with 16 hr light/8hr dark at 20° C. and 80% humidity. Flats were watered with 1 L of tapwater every three days. Five day old seedlings were treated as describedabove with 2 L of either a control (100 mM mannitol pH 6.5) solution or1 L of an experimental (50 mM ammonium nitrate, pH 6.8) solution.Fifteen shoots per time point per treatment were harvested 10, 90 and180 minutes after treatment, flash frozen in liquid nitrogen and storedat −80° C.

Alternatively, seeds of Arabidopsis thaliana (ecotype Wassilewskija)were left at 4° C. for 3 days to vernalize. They were then sown onvermiculite in a growth chamber having 16 hours light/8 hours dark,12,000-14,000 LUX, 70% humidity, and 20° C. They were bottom-wateredwith tap water, twice weekly. Twenty-four days old plants were sprayedwith either water (control) or 0.6% ammonium nitrate at 4 μL/cm² of traysurface. Total shoots and some primary roots were cleaned ofvermiculite, flash-frozen in liquid nitrogen and stored at −80° C.

(n) Methyl Jasmonate

Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were sown in traysand left at 4° C. for 4 days to vernalize before being transferred to agrowth chamber having 16 hr light/8 hr. dark, 13,000 LUX, 70% humidity,20° C. temperature and watered twice a week with 1 L of a 1× Hoagland'ssolution. Approximately 1,000 14 day old plants were spayed with 200-250mls of 0.001% methyl jasmonate in a 0.02% solution of the detergentSilwet L-77. At 1 hr and 6 hrs after treatment, whole, seedlings,including roots, were harvested within a 15 to 20 minute time period,flash-frozen in liquid nitrogen and stored at −80° C.

Seeds of maize hybrid 35A (Pioneer) were sown in water-moistened sand inflats (10 rows, 5-6 seed/row) and covered with clear, plastic lidsbefore being placed in a growth chamber having 16 hr light (25° C.)/8 hrdark (20° C.), 75% relative humidity and 13,000-14,000 LUX. Coveredflats were watered every three days for 7 days. Seedlings were carefullyremoved from the sand and placed in 1-liter beakers with 0.001% methyljasmonate for treatment. Control plants were treated with water. After24 hr, aerial and root tissues were separated and flash frozen in liquidnitrogen prior to storage at −80° C.

(o) Salicylic Acid

Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were sown in traysand left at 4° C. for 4 days to vernalize before being transferred to agrowth chamber having 16 hr light/8 hr. dark, 13,000 LUX, 70% humidity,20° C. temperature and watered twice a week with 1 L of a 1× Hoagland'ssolution. Approximately 1,000 14 day old plants were spayed with 200-250mls of 5 mM salicylic acid (solubilized in 70% ethanol) in a 0.02%solution of the detergent Silwet L-77. At 1 hr and 6 hrs aftertreatment, whole seedlings, including roots, were harvested within a 15to 20 minute time period flash-frozen in liquid nitrogen and stored at−80° C.

Alternatively, seeds of wild-type Arabidopsis thaliana (ecotypeColumbia) and mutant CS3726 were sown in soil type 200 mixed withosmocote fertilizer and Marathon insecticide and left at 4° C. for 3days to vernalize. Flats were incubated at room temperature withcontinuous light. Sixteen days post germination plants were sprayed with2 mM SA, 0.02% SilwettL-77 or control solution (0.02% SilwettL-77.Aerial parts or flowers were harvested 1 hr (EXPT REP: 108471 and108472), 4 hr (EXPT REP: 108469 and 108470), 6 hr (EXPT REP: 108440), 24hr (EXPT REP: 108443, 107953 and 107960) and 3 weeks (EXPT REP: 108475,108476) post-treatment flash frozen and stored at −80° C.

Seeds of maize hybrid 35A (Pioneer) were sown in water-moistened sand inflats (10 rows, 5-6 seed/row) and covered with clear, plastic lidsbefore being placed in a growth chamber having 16 hr light (25° C.)/8 hrdark (20° C.), 75% relative humidity and 13,000-14,000 LUX. Coveredflats were watered every three days for 7 days. Seedlings were carefullyremoved from the sand and placed in 1-liter beakers with 2 mM SA fortreatment. Control plants were treated with water. After 12 hr and 24hr, aerial and root tissues were separated and flash frozen in liquidnitrogen prior to storage at −80° C.

(p) Wounding

Seeds of Arabidopsis thaliana (Wassilewskija) were sown in trays andleft at 4° C. for three days to vernalize before being transferred to agrowth chamber having 16 hr light/8 hr dark, 12,000-14,000 LUX, 70%humidity and 20° C. After 14 days, the leaves were wounded with forceps.Aerial tissues were harvested 1 hour and 6 hours after wounding. Aerialtissues from unwounded plants served as controls. Tissues wereflash-frozen in liquid nitrogen and stored at −80° C.

Seeds of maize hybrid 35A (Pioneer) were sown in water-moistened sand inflats (10 rows, 5-6 seed/row) and covered with clear, plastic lidsbefore being placed in a growth chamber having 16 hr light (25° C.)/8 hrdark (20° C.), 75% relative humidity and 13,000-14,000 LUX. Coveredflats were watered every three days for 7 days. Seedlings were wounded(one leaf nicked by scissors) and placed in 1-liter beakers of water fortreatment. Control plants were treated not wounded. After 1 hr and 6 hraerial and root tissues were separated and flash frozen in liquidnitrogen prior to storage at −80° C.

(q) Drought Stress

Seeds of Arabidopsis thaliana (Wassilewskija) were sown in pots and leftat 4° C. for three days to vernalize before being transferred to agrowth chamber having 16 hr light/8 hr dark, 150,000-160,000 LUX, 20° C.and 70% humidity. After 14 days, aerial tissues were cut and left to dryon 3MM Whatman paper in a petri-plate for 1 hour and 6 hours. Aerialtissues exposed for 1 hour and 6 hours to 3 MM Whatman paper wetted with1× Hoagland's solution served as controls. Tissues were harvested,flash-frozen in liquid nitrogen and stored at −80° C.

Alternatively, Arabidopsis thaliana (Ws) seed was vernalized at 4° C.for 3 days before sowing in Metromix soil type 350. Flats were placed ina growth chamber with 23° C., 16 hr light/8 hr. dark, 80% relativehumidity, ˜13,000 LUX for germination and growth. Plants were wateredwith 1-1.5 L of water every four days. Watering was stopped 16 daysafter germination for the treated samples, but continued for the controlsamples. Rosette leaves and stems (EXPT REP 108477, 108482 and 108483),flowers (see EXPT REP: 108473, 108474) and siliques were harvested 2 d,3 d, 4 d, 5 d, 6 d and 7 d (EXPT REP: 108473) after watering wasstopped. Tissue was flash frozen in liquid nitrogen and kept at −80° C.until RNA was isolated. Flowers and siliques were also harvested on day8 from plants that had undergone a 7 d drought treatment followed by 1day of watering (EXPT REP: 108474). Control plants (whole plants) wereharvested after 5 weeks, flash frozen in liquid nitrogen and stored asabove.

Seeds of maize hybrid 35A (Pioneer) were sown in water-moistened sand inflats (10 rows, 5-6 seed/row) and covered with clear, plastic lidsbefore being placed in a growth chamber having 16 hr light (25° C.)/8 hrdark (20° C.), 75% relative humidity and 13,000-14,000 LUX. Coveredflats were watered every three days for 7 days. Seedlings were carefullyremoved from the sand and placed in empty 1-liter beakers at roomtemperature for treatment. Control plants were placed in water. After 1hr, 6 hr, 12 hr and 24 hr aerial and root tissues were separated andflash frozen in liquid nitrogen prior to storage at −80° C.

(r) Osmotic Stress

Seeds of Arabidopsis thaliana (Wassilewskija) were sown in trays andleft at 4° C. for three days to vernalize before being transferred to agrowth chamber having 16 hr light/8 hr dark, 12,000-14,000 LUX, 20° C.,and 70% humidity. After 14 days, the aerial tissues were cut and placedon 3 MM Whatman paper in a petri-plate wetted with 20% PEG (polyethyleneglycol-M_(r) 8,000) in 1× Hoagland's solution. Aerial tissues on 3 MMWhatman paper containing 1× Hoagland's solution alone served as thecontrol. Aerial tissues were harvested at 1 hour and 6 hours aftertreatment, flash-frozen in liquid nitrogen and stored at −80° C.

Seeds of maize hybrid 35A (Pioneer) were sown in water-moistened sand inflats (10 rows, 5-6 seed/row) and covered with clear, plastic lidsbefore being placed in a growth chamber having 16 hr light (25° C.)/8 hrdark (20° C.), 75% relative humidity and 13,000-14,000 LUX. Coveredflats were watered every three days for 7 days. Seedlings were carefullyremoved from the sand and placed in 1-liter beakers with 20% PEG(polyethylene glycol-M_(r) 8,000) for treatment. Control plants weretreated with water. After 1 hr and 6 hr aerial and root tissues wereseparated and flash frozen in liquid nitrogen prior to storage at −80°C.

Seeds of maize hybrid 35A (Pioneer) were sown in water-moistened sand inflats (10 rows, 5-6 seed/row) and covered with clear, plastic lidsbefore being placed in a growth chamber having 16 hr light (25° C.)/8 hrdark (20° C.), 75% relative humidity and 13,000-14,000 LUX. Coveredflats were watered every three days for 7 days. Seedlings were carefullyremoved from the sand and placed in 1-liter beakers with 150 mM NaCl fortreatment. Control plants were treated with water. After 1 hr, 6 hr, and24 hr aerial and root tissues were separated and flash frozen in liquidnitrogen prior to storage at −80° C.

(s) Heat Shock Treatment

Seeds of Arabidopsis thaliana (Wassilewskija) were sown in trays andleft at 4° C. for three days to vernalize before being transferred to agrowth chamber with 16 hr light/8 hr dark, 12,000-14,000 LUX, 70%humidity and 20° C., fourteen day old plants were transferred to a 42°C. growth chamber and aerial tissues were harvested 1 hr and 6 hr aftertransfer. Control plants were left at 20° C. and aerial tissues wereharvested. Tissues were flashfrozen in liquid nitrogen and stored at−80° C.

Seeds of maize hybrid 35A (Pioneer) were sown in water-moistened sand inflats (10 rows, 5-6 seed/row) and covered with clear, plastic lidsbefore being placed in a growth chamber having 16 hr light (25° C.)/8 hrdark (20° C.), 75% relative humidity and 13,000-14,000 LUX. Coveredflats were watered every three days for 7 days. Seedlings were carefullyremoved from the sand and placed in 1-liter beakers containing 42° C.water for treatment. Control plants were treated with water at 25° C.After 1 hr and 6 hr aerial and root tissues were separated and flashfrozen in liquid nitrogen prior to storage at −80° C.

(t) Cold Shock Treatment

Seeds of Arabidopsis thaliana (Wassilewskija) were sown in trays andleft at 4° C. for three days to vernalize before being transferred to agrowth chamber having 16 hr light/8 hr dark, 12,000-14,000 LUX, 20° C.and 70% humidity. Fourteen day old plants were transferred to a 4° C.dark growth chamber and aerial tissues were harvested 1 hour and 6 hourslater. Control plants were maintained at 20° C. and covered with foil toavoid exposure to light. Tissues were flash-frozen in liquid nitrogenand stored at −80° C.

Seeds of maize hybrid 35A (Pioneer) were sown in water-moistened sand inflats (10 rows, 5-6 seed/row) and covered with clear, plastic lidsbefore being placed in a growth chamber having 16 hr light (25° C.)/8 hrdark (20° C.), 75% relative humidity and 13,000-14,000 LUX. Coveredflats were watered every three days for 7 days. Seedlings were carefullyremoved from the sand and placed in 1-liter beakers containing 4° C.water for treatment. Control plants were treated with water at 25° C.After 1 hr and 6 hr aerial and root tissues were separated and flashfrozen in liquid nitrogen prior to storage at −80° C.

(u) Oxidative Stress—Hydrogen Peroxide Treatment

Seeds of Arabidopsis thaliana (Wassilewskija) were sown in trays andleft at 4° C. for three days to vernalize. Before being transferred to agrowth chamber having 16 hr light/8 hr dark, 12,000-14,000 LUX, 20° C.and 70% humidity. Fourteen day old plants were sprayed with 5 mM H₂O₂(hydrogen peroxide) in a 0.02% Silwett L-77 solution. Control plantswere sprayed with a 0.02% Silwett L-77 solution. Aerial tissues wereharvested 1 hour and 6 hours after spraying, flash-frozen in liquidnitrogen and stored at −80° C.

Seeds of maize hybrid 35A (Pioneer) were sown in water-moistened sand inflats (10 rows, 5-6 seed/row) and covered with clear, plastic lidsbefore being placed in a growth chamber having 16 hr light (25° C.)/8 hrdark (20° C.), 75% relative humidity and 13,000-14,000 LUX. Coveredflats were watered every three days for 7 days. Seedlings were carefullyremoved from the sand and placed in 1-liter beakers with 5 mM H₂O₂ fortreatment. Control plants were treated with water. After 1 hr, 6 hr and24 hr, aerial and root tissues were separated and flash frozen in liquidnitrogen prior to storage at −80° C.

(v) Nitric Oxide Treatment

Seeds of Arabidopsis thaliana (Wassilewskija) were sown in trays andleft at 4° C. for three days to vernalize before being transferred to agrowth chamber having 16 hr light/8 hr dark, 12,000-14,000 LUX, 20° C.and 70% humidity. Fourteen day old plants were sprayed with 5 mM sodiumnitroprusside in a 0.02% Silwett L-77 solution. Control plants weresprayed with a 0.02% Silwett L-77 solution. Aerial tissues wereharvested 1 hour and 6 hours after spraying, flash-frozen in liquidnitrogen and stored at −80° C.

Seeds of maize hybrid 35A (Pioneer) were sown in water-moistened sand inflats (10 rows, 5-6 seed/row) and covered with clear, plastic lidsbefore being placed in a growth chamber having 16 hr light (25° C.)/8 hrdark (20° C.), 75% relative humidity and 13,000-14,000 LUX. Coveredflats were watered every three days for 7 days. Seedlings were carefullyremoved from the sand and placed in 1-liter beakers with 5 mMnitroprusside for treatment. Control plants were treated with water.After 1 hr, 6 hr and 12 hr, aerial and root tissues were separated andflash frozen in liquid nitrogen prior to storage at −80° C.

(w) S4 Immature Buds, Inflorescence Meristem

Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were sown in potsand left at 4° C. for two to three days to vernalize. They were thentransferred to a growth chamber. Plants were grown under long-day (16 hrlight: 8 hr dark) conditions, 7000-8000 LUX light intensity, 70%humidity, and 22° C. temperature. Inflorescences containing immaturefloral buds [stages 1-12; Smyth et al., 1990] as well as theinflorescence meristem were harvested and flash frozen in liquidnitrogen.

(x) S5 Flowers (Opened)

Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were sown in potsand left at 4° C. for two to three days to vernalize. They were thentransferred to a growth chamber. Plants were grown under long-day (16 hrlight: 8 hr dark) conditions, 7000-8000 LUX light intensity, 70%humidity, and 22° C. temperature. Mature, unpollinated flowers [stages12-14; Smyth et al. 1990] were harvested and flash frozen in liquidnitrogen.

(y) S6 Siliques (All Stages)

Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were sown in potsand left at 4° C. for two to three days to vernalize. They were thentransferred to a growth chamber. Plants were grown under long-day (16 hrlight: 8 hr dark) conditions, 7000-8000 LUX light intensity, 70%humidity, and 22° C. temperature. Siliques bearing developing seedscontaining post fertilization through pre-heart stage [0-72 hours afterfertilization (HAF)], heart-through early curled cotyledon stage [72-120HAF] and late-curled cotyledon stage [>120 HAF] embryos were harvestedseparately and pooled prior to RNA isolation in a mass ratio of 1:1:1.The tissues were then flash frozen in liquid nitrogen. Description ofthe stages of Arabidopsis embryogenesis used were reviewed by Bowman(1994).

(z) Arabidopsis Endosperm

mea/mea Fruits 0-10 mm

Seeds of Arabidopsis thaliana heterozygous for thefertilization-independent endosperm1 (fie1) [Ohad et al., 1996; ecotypeLandsberg erecta (Ler)] were sown in pots and left at 4° C. for two tothree days to vernalize. Kiyosue et al. (1999) subsequently determinedthat fie1 was allelic to the gametophytic maternal effect mutant medea(Grossniklaus et al., 1998). Imbibed seeds were then transferred to agrowth chamber. Plants were grown under long-day (16 hr light: 8 hrdark) conditions, 7000-8000 LUX light intensity, 70% humidity, and 22°C. temperature. 1-2 siliques (fruits) bearing developing seeds justprior to dessication [9 days after flowering (DAF)] were selected fromeach plant and were hand-dissected to identify wild-type, mea/+heterozygotes, and mea/mea homozygous mutant plants. At this stage,homozygous mea/mea plants produce short siliques that contain >70%aborted seed and can be distinguished from those produced by wild-type(100% viable seed) and mea/+ heterozygous (50% viable seed) plants (Ohadet al., 1996; Grossniklaus et al., 1998; Kiyosue et al., 1999). Siliques0-10 mm in length containing developing seeds 0-9 DAF produced byhomozygous mea/mea plants were harvested and flash frozen in liquidnitrogen.

Pods 0-10 mm (Control Tissue for Sample 70)

Seeds of Arabidopsis thaliana heterozygous for thefertilization-independent endosperm1 (fie1) [Ohad et al., 1996; ecotypeLandsberg erecta (Ler)] were sown in pots and left at 4° C. for two tothree days to vernalize. Kiyosue et al. (1999) subsequently determinedthat fie1 was allelic to the gametophytic maternal effect mutant medea(Grossniklaus et al., 1998). Imbibed seeds were then transferred to agrowth chamber. Plants were grown under long-day (16 hr light: 8 hrdark) conditions, 7000-8000 LUX light intensity, 70% humidity, and 22°C. temperature. 1-2 siliques (fruits) bearing developing seeds justprior to dessication [9 days after flowering (DAF)] were selected fromeach plant and were hand-dissected to identify wild-type, mea/+heterozygotes, and mea/mea homozygous mutant plants. At this stage,homozygous mea/mea plants produce short siliques that contain >70%aborted seed and can be distinguished from those produced by wild-type(100% viable seed) and mea/+ heterozygous (50% viable seed) plants (Ohadet al., 1996; Grossniklaus et al., 1998; Kiyosue et al., 1999). Siliques0-10 mm in length containing developing seeds 0-9 DAF produced bysegregating wild-type plants were opened and the seeds removed. Theremaining tissues (pods minus seed) were harvested and flash frozen inliquid nitrogen.

(aa) Arabidopsis Seeds

Fruits (Pod+Seed) 0-5 mm

Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were sown in potsand left at 4° C. for two to three days to vernalize. They were thentransferred to a growth chamber. Plants were grown under long-day (16 hrlight: 8 hr dark) conditions, 7000-8000 LUX light intensity, 70%humidity, and 22° C. temperature. 3-4 siliques (fruits) bearingdeveloping seeds were selected from at least 3 plants and werehand-dissected to determine what developmental stage(s) were representedby the enclosed embryos. Description of the stages of Arabidopsisembryogenesis used in this determination were summarized by Bowman(1994). Silique lengths were then determined and used as an approximatedeterminant for embryonic stage. Siliques 0-5 mm in length containingpost fertilization through pre-heart stage [0-72 hours afterfertilization (HAF)] embryos were harvested and flash frozen in liquidnitrogen.

Fruits (Pod+Seed) 5-10 mm

Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were sown in potsand left at 4° C. for two to three days to vernalize. They were thentransferred to a growth chamber. Plants were grown under long-day (16 hrlight: 8 hr dark) conditions, 7000-8000 LUX light intensity, 70%humidity, and 22° C. temperature. 3-4 siliques (fruits) bearingdeveloping seeds were selected from at least 3 plants and werehand-dissected to determine what developmental stage(s) were representedby the enclosed embryos. Description of the stages of Arabidopsisembryogenesis used in this determination were summarized by Bowman(1994). Silique lengths were then determined and used as an approximatedeterminant for embryonic stage. Siliques 5-10 mm in length containingheart-through early upturned-U-stage [72-120 hours after fertilization(HAF)] embryos were harvested and flash frozen in liquid nitrogen.

Fruits (Pod+Seed)>10 mm

Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were sown in potsand left at 4° C. for two to three days to vernalize. They were thentransferred to a growth chamber. Plants were grown under long-day (16 hrlight: 8 hr dark) conditions, 7000-8000 LUX light intensity, 70%humidity, and 22° C. temperature. 3-4 siliques (fruits) bearingdeveloping seeds were selected from at least 3 plants and werehand-dissected to determine what developmental stage(s) were representedby the enclosed embryos. Description of the stages of Arabidopsisembryogenesis used in this determination were summarized by Bowman(1994). Silique lengths were then determined and used as an approximatedeterminant for embryonic stage. Siliques >10 mm in length containinggreen, late upturned-U-stage [>120 hours after fertilization (HAF)-9days after flowering (DAF)] embryos were harvested and flash frozen inliquid nitrogen.

Green Pods 5-10 mm (Control Tissue for Samples 72-74)

Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were sown in potsand left at 4° C. for two to three days to vernalize. They were thentransferred to a growth chamber. Plants were grown under long-day (16 hrlight: 8 hr dark) conditions, 7000-8000 LUX light intensity, 70%humidity, and 22° C. temperature. 3-4 siliques (fruits) bearingdeveloping seeds were selected from at least 3 plants and werehand-dissected to determine what developmental stage(s) were representedby the enclosed embryos. Description of the stages of Arabidopsisembryogenesis used in this determination were summarized by Bowman(1994). Silique lengths were then determined and used as an approximatedeterminant for embryonic stage. Green siliques 5-10 mm in lengthcontaining developing seeds 72-120 hours after fertilization (HAF)] wereopened and the seeds removed. The remaining tissues (green pods minusseed) were harvested and flash frozen in liquid nitrogen.

Green Seeds from Fruits >10 mm

Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were sown in potsand left at 4° C. for two to three days to vernalize. They were thentransferred to a growth chamber. Plants were grown under long-day (16 hrlight: 8 hr dark) conditions, 7000-8000 LUX light intensity, 70%humidity, and 22° C. temperature. 3-4 siliques (fruits) bearingdeveloping seeds were selected from at least 3 plants and werehand-dissected to determine what developmental stage(s) were representedby the enclosed embryos. Description of the stages of Arabidopsisembryogenesis used in this determination were summarized by Bowman(1994). Silique lengths were then determined and used as an approximatedeterminant for embryonic stage. Green siliques >10 mm in lengthcontaining developing seeds up to 9 days after flowering (DAF)] wereopened and the seeds removed and harvested and flash frozen in liquidnitrogen.

Brown Seeds from Fruits >10 mm

Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were sown in potsand left at 4° C. for two to three days to vernalize. They were thentransferred to a growth chamber. Plants were grown under long-day (16 hrlight: 8 hr dark) conditions, 7000-8000 LUX light intensity, 70%humidity, and 22° C. temperature. 3-4 siliques (fruits) bearingdeveloping seeds were selected from at least 3 plants and werehand-dissected to determine what developmental stage(s) were representedby the enclosed embryos. Description of the stages of Arabidopsisembryogenesis used in this determination were summarized by Bowman(1994). Silique lengths were then determined and used as an approximatedeterminant for embryonic stage. Yellowing siliques >10 mm in lengthcontaining brown, dessicating seeds >11 days after flowering (DAF)] wereopened and the seeds removed and harvested and flash frozen in liquidnitrogen.

Green/Brown Seeds from Fruits >10 mm

Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were sown in potsand left at 4° C. for two to three days to vernalize. They were thentransferred to a growth chamber. Plants were grown under long-day (16 hrlight: 8 hr dark) conditions, 7000-8000 LUX light intensity, 70%humidity, and 22° C. temperature. 3-4 siliques (fruits) bearingdeveloping seeds were selected from at least 3 plants and werehand-dissected to determine what developmental stage(s) were representedby the enclosed embryos. Description of the stages of Arabidopsisembryogenesis used in this determination were summarized by Bowman(1994). Silique lengths were then determined and used as an approximatedeterminant for embryonic stage. Green siliques >10 mm in lengthcontaining both green and brown seeds >9 days after flowering (DAF)]were opened and the seeds removed and harvested and flash frozen inliquid nitrogen.

Mature Seeds (24 Hours After Imbibition)

Mature dry seeds of Arabidopsis thaliana (ecotype Wassilewskija) weresown onto moistened filter paper and left at 4° C. for two to three daysto vernalize. Imbibed seeds were then transferred to a growth chamber[16 hr light: 8 hr dark conditions, 7000-8000 LUX light intensity, 70%humidity, and 22° C. temperature], the emerging seedlings harvestedafter 48 hours and flash frozen in liquid nitrogen.

Mature Seeds (Dry)

Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were sown in potsand left at 4° C. for two to three days to vernalize. They were thentransferred to a growth chamber. Plants were grown under long-day (16 hrlight: 8 hr dark) conditions, 7000-8000 LUX light intensity, 70%humidity, and 22° C. temperature and taken to maturity. Mature dry seedsare collected, dried for one week at 28° C., and vernalized for one weekat 4° C. before used as a source of RNA.

Ovules

Seeds of Arabidopsis thaliana heterozygous for pistillata (pi) [ecotypeLandsberg erecta (Ler)] were sown in pots and left at 4° C. for two tothree days to vernalize. They were then transferred to a growth chamber.Plants were grown under long-day (16 hr light: 8 hr dark) conditions,7000-8000 LUX light intensity, 76% humidity, and 24° C. temperature.Inflorescences were harvested from seedlings about 40 days old. Theinflorescences were cut into small pieces and incubated in the followingenzyme solution (pH 5) at room temperature for 0.5-1 hr.: 0.2%pectolyase Y-23, 0.04% pectinase, 5 mM MES, 3% Sucrose and MS salts(1900 mg/l KNO₃, 1650 mg/l NH₄NO₃, 370 mg/l MgSO₄.7H₂O, 170 mg/l KH₂PO₄,440 mg/l CaCl₂.2H₂O, 6.2 mg/l H₂BO₃, 15.6 mg/l MnSO₄.4H₂O, 8.6 mg/lZnSO₄.7H₂O, 0.25 mg/l NaMoO₄.2H₂O, 0.025 mg/l CuCO₄.5H₂O, 0.025 mg/lCoCl₂.6H₂O, 0.83 mg/l KI, 27.8 mg/l FeSO₄.7H₂O, 37.3 mg/l Disodium EDTA,pH 5.8). At the end of the incubation the mixture of inflorescencematerial and enzyme solution was passed through a size 60 sieve and thenthrough a sieve with a pore size of 125 μm. Ovules greater than 125 μmin diameter were collected, rinsed twice in B5 liquid medium (2500 mg/lKNO₃, 250 mg/l MgSO₄.7H₂O, 150 mg/l NaH2PO₄.H₂O, 150 mg/l CaCl₂.2H₂O,134 mg/l (NH₄)₂ CaCl₂.SO₄, 3 mg/l H₂BO₃, 10 mg/l MnSO₄.4H₂O, 2ZnSO₄.7H₂O, 0.25 mg/l NaMoO₄.2H₂O, 0.025 mg/l CuCO₄.5H₂O, 0.025 mg/lCoCl₂.6H₂O, 0.75 mg/l KI, 40 mg/l EDTA sodium ferric salt, 20 g/lsucrose, 10 mg/l Thiamine hydrochloride, 1 mg/l Pyridoxinehydrochloride, 1 mg/l Nicotinic acid, 100 mg/l myo-inositol, pH 5.5)),rinsed once in deionized water and flash frozen in liquid nitrogen. Thesupernatant from the 125 μm sieving was passed through subsequent sievesof 50 μm and 32 μm. The tissue retained in the 32 μm sieve was collectedand mRNA prepared for use as a control.

(bb) Herbicide Treatment

Arabidopsis thaliana (Ws) seeds were sterilized for 5 min. with 30%bleach, 50 μl Triton in a total volume of 50 ml. Seeds were vernalizedat 4° C. for 3 days before being plated onto GM agar plates at a densityof about 144 seeds per plate. Plates were incubated in a Percival growthchamber having 16 hr light/8 hr dark, 80% relative humidity, 22° C. and11,000 LUX for 14 days.

Plates were sprayed (˜0.5 mls/plate) with water, Finale (1.128 g/L),Glean (1.88 g/L), RoundUp (0.01 g/L) or Trimec (0.08 g/L). Tissue wascollected and flash frozen in liquid nitrogen at the following timepoints: 0, 1, 2, 4 (EXPT REP: 107871 (Finale), 107881 (Glean), 107896(Round-up) and 107886 (Trimec)), 8, 12 (EXPT REP: 108467 (Finale),108468 (Glean), 108465 (Round-up) and 108466, 107891 (Trimec)), and 24hours. Frozen tissue was stored at −80° C. prior to RNA isolation.

(cc) Ap2

Seeds of Arabidopsis thaliana (ecotype Landesberg erecta) and floralmutant apetala2 (Jofuku et al., 1994, Plant Cell 6:1211-1225) were sownin pots and left at 4° C. for two to three days to vernalize. They werethen transferred to a growth chamber. Plants were grown under long-day(16 hr light, 8 hr dark) conditions 7000-8000 LUX light intensity, 70%humidity and 22° C. temperature. Inflorescences containing immaturefloral buds (stages 1-7; Bowman, 1994) as wel as the inflorescencemeristem were harvested and flashfrozen. Polysomal polyA+ RNA wasisolated from tissue according to Cox and Goldberg, 1988).

(dd) Protein Degradation

Arabidopsis thaliana (ecotype Ws) wild-type and 13B12-1 (homozygous)mutant seed were sown in pots containing Metro-mix 350 soil andincubated at 4° C. for four days. Vernalized seeds were germinated inthe greenhouse (16 hr light/8 hr dark) over a 7 day period. Mutantseedlings were sprayed with 0.02% (active ingredient) Finale to confirmtheir transgenic standing. Plants were grown until the mutant phenotype(either multiple pistils in a single flower and/or multiple branchingper node) was apparent. Young inflorescences immediately forming fromthe multiple-branched stems were cut and flash frozen in liquidnitrogen. Young inflorescences from wild-type plants grown in paralleland under identical conditions were collected as controls. All collectedtissue was stored at −80° C. until RNA isolation. (EXPT REP 108451)

(ee) Root Tips

Seeds of Arabidopsis thaliana (ecotye Ws) were placed on MS plates andvernalized at 4° C. for 3 days before being placed in a 25° C. growthchamber having 16 hr light/8 hr dark, 70% relative humidity and about 3W/m². After 6 days, young seedlings were transferred to flaskscontaining B5 liquid medium, 1% sucrose and 0.05 mg/l indole-3-butyricacid. Flasks were incubated at room temperature with 100 rpm agitation.Media was replaced weekly. After three weeks, roots were harvested andincubated for 1 hr with 2% pectinase, 0.2% cellulase, pH 7 beforestraining through a #80 (Sigma) sieve. The root body material remainingon the sieve (used as the control) was flash frozen and stored at −80°C. until use. The material that passed through the #80 sieve wasstrained through a #200 (Sigma) sieve and the material remaining on thesieve (root tips) was flash frozen and stored at −80° C. until use.Approximately 10 mg of root tips were collected from one flask of rootculture.

Seeds of maize hybrid 35A (Pioneer) were sown in water-moistened sand inflats (10 rows, 5-6 seed/row) and covered with clear, plastic lidsbefore being placed in a growth chamber having 16 hr light (25° C.)/8 hrdark (20° C.), 75% relative humidity and 13,000-14,000 LUX. Coveredflats were watered every three days for 8 days. Seedlings were carefullyremoved from the sand and the root tips (˜2 mm long) were removed andflash frozen in liquid nitrogen prior to storage at −80° C. The tissuesabove the root tips (˜1 cm long) were cut, treated as above and used ascontrol tissue.

(ff) rt1

The rt1 allele is a variation of rt1 rootless1 and is recessive. Plantsdisplaying the rt1 phenotype have few or no secondary roots.

Seed from plants segregating for rt1 were sown on sand and placed in agrowth chamber having 16 hr light/8 hr dark, 13,000 LUX, 70% humidityand 20° C. temperature. Plants were watered every three days with tapwater. Eleven (11) day old seedlings were carefully removed from thesand, keeping the roots intact. rt1-type seedlings were separated fromtheir wild-type counterparts and the root tissue isolated. Root tissuefrom normal seedlings (control) and rtl mutants were flash frozen inliquid nitrogen and stored at −80° C. until use.

(gg) Imbibed Seed

Seeds of maize hybrid 35A (Pioneer) were sown in water-moistened sand incovered flats (10 rows, 5-6 seed/row) and covered with clear, plasticlids before being placed in a growth chamber having 16 hr light (25°C.)/8 hr dark (20° C.), 75% relative humidity and 13,000-14,000 LUX. Oneday after sowing, whole seeds were flash frozen in liquid nitrogen priorto storage at −80° C. Two days after sowing, embryos and endosperm wereisolated and flash frozen in liquid nitrogen prior to storage at −80° C.On days 3-6, aerial tissues, roots and endosperm were isolated and flashfrozen in liquid nitrogen prior to storage at −80° C.

(hh) Rough Sheath2-R (rs2-R) Mutants (1400-6/S-17)

This experiment was conducted to identify abnormally expressed genes inthe shoot apex of rough sheath2-R (rs2-R) mutant plants. rs2 encodes amyb domain DNA binding protein that functions in repression of severalshoot apical meristem expressed homeobox genes. Two homeobox genetargets are known for rs2 repression, rough sheath1, liguleless 3. Therecessive loss of function phenotype of rs2-R homozygous plants isdescribed in Schneeberger et al. 1998 Development 125: 2857-2865.

The seed stock genetically segregates 1:1 for rs2-R/rs2-R:rs2-R/+

Preparation of tissue samples: 160 seedlings pooled from 2 and 3 weekold plants grown in sand. Growth conditions; Conviron #107 @ 12 hrdays/12 hr night, 25° C., 75% humidity. Shoot apex was dissected toinclude leaf three and older. (Pictures available upon request).

1) rough sheath2-R homozygous (mutant) shoot apex2) rough sheath2-R heterozygous (wt, control) shoot apex

(ii) Leaf Mutant 3642:

Mutant 3642 is a recessive mutation that causes abnormal leafdevelopment. The leaves of mutant 3642 plants are characterized by leaftwisting and irregular leaf shape. Mutant 3642 plants also exhibitabnormally shaped floral organs which results in reduced fertility.

Seed segregating for the mutant phenotype was sown in Metro-mix 350 soiland grown in a Conviron growth chamber with watering by sub-irrigationtwice a week. Environmental conditions were set at 20 degrees Celsius,70% humidity with an 8 hour day, 16 hour night light regime. Plants wereharvested after 4 weeks of growth and the entire aerial portion of theplant was harvested and immediately frozen in liquid nitrogen and storedat −80 C. Mutant phenotype plants were harvested separately from normalphenotype plants, which serve as the control tissue.

(jj) Flowers (Green, White or Buds)

Approximately 10 μl of Arabidopsis thaliana seeds (ecotype Ws) were sownon 350 soil (containing 0.03% marathon) and vernalized at 4 C for 3days. Plants were then grown at room temperature under fluorescentlighting until flowering. Flowers were harvested after 28 days in threedifferent categories. Buds that had not opened at all and werecompletely green were categorized as “flower buds” (also referred to asgreen buds by the investigator). Buds that had started to open, withwhite petals emerging slightly were categorized as “green flowers” (alsoreferred to as white buds by the investigator). Flowers that had openedmostly (with no silique elongation) with white petals completely visiblewere categorized as “white flowers” (also referred to as open flowers bythe investigator). Buds and flowers were harvested with forceps, flashfrozen in liquid nitrogen and stored at −80 C until RNA was isolated.

2. Microarray Hybridization Procedures

Microarray technology provides the ability to monitor mRNA transcriptlevels of thousands of genes in a single experiment. These experimentssimultaneously hybridize two differentially labeled fluorescent cDNApools to glass slides that have been previously spotted with cDNA clonesof the same species. Each arrayed cDNA spot will have a correspondingratio of fluorescence that represents the level of disparity between therespective mRNA species in the two sample pools. Thousands ofpolynucleotides can be spotted on one slide, and each experimentgenerates a global expression pattern.

Coating Slides

The microarray consists of a chemically coated microscope slide,referred herein as a “chip” with numerous polynucleotide samples arrayedat a high density. The poly-L-lysine coating allows for this spotting athigh density by providing a hydrophobic surface, reducing the spreadingof spots of DNA solution arrayed on the slides. Glass microscope slides(Gold Seal #3010 manufactured by Gold Seal Products, Portsmouth, N.H.,USA) were coated with a 0.1% W/V solution of Poly-L-lysine (Sigma, St.Louis, Mo.) using the following protocol:

1. Slides were placed in slide racks (Shandon Lipshaw #121). The rackswere then put in chambers (Shandon Lipshaw #121).2. Cleaning solution was prepared:

70 g NaOH was dissolved in 280 mL ddH₂O.

420 mL 95% ethanol was added. The total volume was 700 mL (=2×350 mL);it was stirred until completely mixed.

If the solution remained cloudy, ddH₂O was added until clear.

3. The solution was poured into chambers with slides; the chambers werecovered with glass lids. The solution was mixed on an orbital shaker for2 hr.4. The racks were quickly transferred to fresh chambers filled withddH₂O. They were rinsed vigorously by plunging racks up and down.

Rinses were repeated 4× with fresh ddH₂O each time, to remove all tracesof NaOH-ethanol.

5. Polylysine solution was prepared:

0 mL poly-L-lysine+70 mL tissue culture PBS in 560 mL water, usingplastic graduated cylinder and beaker.

6. Slides were transferred to polylysine solution and shaken for 1 hr.7. The rack was transferred to a fresh chambers filled with ddH₂O. Itwas plunged up and down 5× to rinse.8. The slides were centrifuged on microtiter plate carriers (papertowels were placed below the rack to absorb liquid) for 5 min. @ 500rpm. The slide racks were transferred to empty chambers with covers.9. Slide racks were dried in a 45 C oven for 10 min.10. The slides were stored in a closed plastic slide box.11. Normally, the surface of lysine coated slides was not veryhydrophobic immediately after this process, but became increasinglyhydrophobic with storage. A hydrophobic surface helped ensure that spotsdidn't run together while printing at high densities. After they agedfor 10 days to a month the slides were ready to use. However, coatedslides that have been sitting around for long periods of time wereusually too old to be used. This was because they developed opaquepatches, visible when held to the light, and these resulted in highbackground hybridization from the fluorescent probe.

Alternatively, precoated glass slides were purchased from TeleChemInternation, Inc. (Sunnyvale, Calif., 94089; catalog number SMM-25,Superamine substrates).

PCR Amplification of cDNA Clone Inserts

Polynucleotides were amplified from Arabidopsis cDNA clones using insertspecific probes. The resulting 100 uL PCR reactions were purified withQiaquick 96 PCR purification columns (Qiagen, Valencia, Calif., USA) andeluted in 30 uL of 5 mM Tris. 8.5 uL of the elution were mixed with 1.5uL of 20×SSC to give a final spotting solution of DNA in 3×SSC. Theconcentrations of DNA generated from each clone varied between 10-100ng/ul, but were usually about 50 ng/ul.

Arraying of PCR Products on Glass Slides

PCR products from cDNA clones were spotted onto the poly-L-Lysine coatedglass slides using an arrangement of quill-tip pins (ChipMaker 3spotting pins; Telechem, International, Inc., Sunnyvale, Calif., USA)and a robotic arrayer (PixSys 3500, Cartesian Technologies, Irvine,Calif., USA). Around 0.5 nl of a prepared PCR product was spotted ateach location to produce spots with approximately 100 um diameters. Spotcenter-to-center spacing was from 180 um to 210 um depending on thearray. Printing was conducted in a chamber with relative humidity set at50%.

Slides containing maize sequences were purchased from Agilent Technology(Palo Alto, Calif. 94304).

Post-Processing of Slides

After arraying, slides were processed through a series ofsteps—rehydration, UV cross-linking, blocking and denaturation—requiredprior to hybridization. Slides were rehydrated by placing them over abeaker of warm water (DNA face down), for 2-3 sec, to distribute the DNAmore evenly within the spots, and then snap dried on a hot plate (DNAside, face up). The DNA was then cross-linked to the slides by UVirradiation (60-65 mJ; 2400 Stratalinker, Stratagene, La Jolla, Calif.,USA).

Following this a blocking step was performed to modify remaining freelysine groups, and hence minimize their ability to bind labeled probeDNA. To achieve this the arrays were placed in a slide rack. An emptyslide chamber was left ready on an orbital shaker. The rack was bentslightly inwards in the middle, to ensure the slides would not run intoeach other while shaking. The blocking solution was prepared as follows:

3×350-ml glass chambers (with metal tops) were set to one side, and alarge round Pyrex dish with dH₂O was placed ready in the microwave. Atthis time, 15 ml sodium borate was prepared in a 50 ml conical tube.6-g succinic anhydride was dissolved in approx. 325-350 mL1-methyl-2-pyrrolidinone. Rapid addition of reagent was crucial.a. Immediately after the last flake of the succinic anhydride dissolved,the 15-mL sodium borate was added.b. Immediately after the sodium borate solution mixed in, the solutionwas poured into an empty slide chamber.c. The slide rack was plunged rapidly and evenly in the solution. It wasvigorously shaken up and down for a few seconds, making sure slidesnever left the solution.d. It was mixed on an orbital shaker for 15-20 min. Meanwhile, the waterin the Pyrex dish (enough to cover slide rack) was heated to boiling.

Following this, the slide rack was gently plunge in the 95 C water (juststopped boiling) for 2 min. Then the slide rack was plunged 5× in 95%ethanol. The slides and rack were centrifuged for 5 min. @ 500 rpm. Theslides were loaded quickly and evenly onto the carriers to avoidstreaking. The arrays were used immediately or store in slide box.

The Hybridization process began with the isolation of mRNA from the twotissues (see “Isolation of total RNA” and “Isolation of mRNA”, below) inquestion followed by their conversion to single stranded cDNA (see“Generation of probes for hybridization”, below). The cDNA from eachtissue was independently labeled with a different fluorescent dye andthen both samples were pooled together. This final differentiallylabeled cDNA pool was then placed on a processed microarray and allowedto hybridize (see “Hybridization and wash conditions”, below).

Isolation of Total RNA

Approximately 1 g of plant tissue was ground in liquid nitrogen to afine powder and transferred into a 50-ml centrifuge tube containing 10ml of Trizol reagent. The tube was vigorously vortexed for 1 min andthen incubated at room temperature for 10-20 min. on an orbital shakerat 220 rpm. Two ml of chloroform was added to the tube and the solutionvortexed vigorously for at least 30-sec before again incubating at roomtemperature with shaking. The sample was then centrifuged at 12,000×g(10,000 rpm) for 15-20 min at 4° C. The aqueous layer was removed andmixed by inversion with 2.5 ml of 1.2 M NaCl/0.8 M Sodium Citrate and2.5 ml of isopropyl alcohol added. After a 10 min. incubation at roomtemperature, the sample was centrifuged at 12,000×g (10,000 rpm) for 15min at 4° C. The pellet was washed with 70% ethanol, re-centrifuged at8,000 rpm for 5 min and then air dried at room temperature for 10 min.The resulting total RNA was dissolved in either TE (10 mM Tris-HCl, 1 mMEDTA, pH 8.0) or DEPC (diethylpyrocarbonate) treated deionized water(RNAse-free water). For subsequent isolation of mRNA using the Qiagenkit, the total RNA pellet was dissolved in RNAse-free water.

Isolation of mRNA

mRNA was isolated using the Qiagen Oligotex mRNA Spin-Column protocol(Qiagen, Valencia, Calif.). Briefly, 500 μl OBB buffer (20 mM Tris-Ci,pH 7.5, 1 M NaCl, 2 mM EDTA, 0.2% SDS) was added to 500 μl of total RNA(0.5-0.75 mg) and mixed thoroughly. The sample was first incubated at70° C. for 3 min, then at room temperature for 10 minutes and finallycentrifuged for 2 min at 14,000-18,000×g. The pellet was resuspended in400 μl OW2 buffer (10 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA) byvortexing, the resulting solution placed on a small spin column in a 1.5ml RNase-free microcentrifuge tube and centrifuged for 1 min at14,000-18,000×g. The spin column was transferred to a new 1.5 mlRNase-free microcentrifuge tube and washed with 400 μl of OW2 buffer. Torelease the isolated mRNA from the resin, the spin column was againtransferred to a new RNase-free 1.5 ml microcentrifuge tube, 20-100 μl70° C. OEB buffer (5 mM Tris-Cl, pH 7.5) added and the resin resuspendedin the resulting solution via pipeting. The mRNA solution was collectedafter centrifuging for 1 min at 14,000-18,000×g.

Alternatively, mRNA was isolated using the Stratagene Poly(A) Quik mRNAIsolation Kit (Startagene, La Jolla, Calif.). Here, up to 0.5 mg oftotal RNA (maximum volume of 1 ml) was incubated at 65° C. for 5minutes, snap cooled on ice and 0.1× volumes of 10× sample buffer (10 mMTris-HCl (pH 7.5), 1 mM EDTA (pH 8.0) 5 M NaCl) added. The RNA samplewas applied to a prepared push column and passed through the column at arate of ˜1 drop every 2 sec. The solution collected was reapplied to thecolumn and collected as above. 200 μl of high salt buffer (10 mMTris-HCl (pH 7.5), 1 mM EDTA, 0.5 NaCl) was applied to the column andpassed through the column at a rate of ˜1 drop every 2 sec. This stepwas repeated and followed by three low salt buffer (10 mM Tris-HCl (pH7.5), 1 mM EDTA, 0.1 M NaCl) washes preformed in a similar manner. mRNAwas eluted by applying to the column four separate 200 μl aliquots ofelution buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA) preheated to 65° C.Here, the elution buffer was passed through the column at a rate of 1drop/sec. The resulting mRNA solution was precipitated by adding 0.1×volumes of 10× sample buffer, 2.5 volumes of ice-cold 100% ethanol,incubating overnight at −20° C. and centrifuging at 14,000-18,000×g for20-30 min at 4° C. The pellet was washed with 70% ethanol and air driedfor 10 min. at room temperature before resuspension in RNase-freedeionized water.

Preparation of Yeast Controls

Plasmid DNA was isolated from the following yeast clones using Qiagenfiltered maxiprep kits (Qiagen, Valencia, Calif.): YAL022c(Fun26),YAL031c(Fun21), YBR032w, YDL131w, YDL182w, YDL194w, YDL196w, YDR050c andYDR116c. Plasmid DNA was linearized with either BsrBI (YAL022c(Fun26),YAL031c(Fun21), YDL131w, YDL182w, YDL194w, YDL196w, YDR050c) or AflIII(YBR032w, YDR116c) and isolated.

In Vitro Transcription of Yeast Clones

The following solution was incubated at 37° C. for 2 hours: 17 μl ofisolated yeast insert DNA (1 μg), 20 μl 5× buffer, 10 μl 100 mM DTT, 2.5μl (100 U) RNasin, 20 μl 2.5 mM (ea.) rNTPs, 2.7 μl (40 U) SP6polymerase and 27.8 μl RNase-free deionized water. 2 μl (2 U) AmpliDNase I was added and the incubation continued for another 15 min. 10 μl5M NH₄OAC and 100 μl phenol:chloroform:isoamyl alcohol (25:24:1) wereadded, the solution vortexed and then centrifuged to separate thephases. To precipitate the RNA, 250 μl ethanol was added and thesolution incubated at −20° C. for at least one hour. The sample was thencentrifuged for 20 min at 4° C. at 14,000-18,000×g, the pellet washedwith 500 μl of 70% ethanol, air dried at room temperature for 10 min andresuspended in 100 μl of RNase-free deionized water. The precipitationprocedure was then repeated.

Alternatively, after the two-hour incubation, the solution was extractedwith phenol/chloroform once before adding 0.1 volume 3M sodium acetateand 2.5 volumes of 100% ethanol. The solution was centrifuged at 15,000rpm, 4° C. for 20 minutes and the pellet resuspended in RNase-freedeionized water. The DNase I treatment was carried out at 37° C. for 30minutes using 2 U of Ampli DNase I in the following reaction condition:50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂. The DNase I reaction was thenstopped with the addition of NH₄OAC and phenol:chloroform:isoamylalcohol (25:24:1), and RNA isolated as described above.

0.15-2.5 ng of the in vitro transcript RNA from each yeast clone wereadded to each plant mRNA sample prior to labeling to serve as positive(internal) probe controls.

Generation of Probes for Hybridization

Generation of Labeled Probes for Hybridization from First-Strand cDNA

Hybridization probes were generated from isolated mRNA using an Atlas™Glass Fluorescent Labeling Kit (Clontech Laboratories, Inc., Palo Alto,Calif., USA). This entails a two step labeling procedure that firstincorporates primary aliphatic amino groups during cDNA synthesis andthen couples fluorescent dye to the cDNA by reaction with the aminofunctional groups. Briefly, 5 μg of oligo(dT)₁₈ primerd(TTTTTTTTTTTTTTTTTTV) was mixed with Poly A+mRNA (1.5-2 μg mRNAisolated using the Qiagen-Oligotex mRNA Spin-Column protocol or theStratagene Poly(A) Quik mRNA Isolation protocol (Stratagene, La Jolla,Calif., USA)) in a total volume of 25 μl. The sample was incubated in athermocycler at 70° C. for 5 min, cooled to 48° C. and 10 μl of 5× cDNASynthesis Buffer (kit supplied), 5 μl 10× dNTP mix (dATP, dCTP, dGTP,dTTP and aminoallyl-dUTP; kit supplied), 7.5 μl deionized water and 2.5μl MMLV Reverse Transcriptase (500 U) added. The reaction was thenincubated at 48° C. for 30 minutes, followed by 1 hr incubation at 42°C. At the end of the incubation the reaction was heated to 70° C. for 10min, cooled to 37° C. and 0.5 μl (5 U) RNase H added, before incubatingfor 15 min at 37° C. The solution was vortexed for 1 min after theaddition of 0.5 μl 0.5 M EDTA and 5 μl of QuickClean Resin (kitsupplied) then centrifuged at 14,000-18,000×g for 1 min. After removingthe supernatant to a 0.45 μm spin filter (kit supplied), the sample wasagain centrifuged at 14,000-18,000×g for 1 min, and 5.5 μl 3 M sodiumacetate and 137.5 μl of 100% ethanol added to the sample beforeincubating at −20° C. for at least 1 hr. The sample was then centrifugedat 14,000-18,000×g at 4° C. for 20 min, the resulting pellet washed with500 μl 70% ethanol, air-dried at room temperature for 10 min andresuspended in 10 μl of 2× fluorescent labeling buffer (kit provided).10 μl each of the fluorescent dyes Cy3 and Cy5 (Amersham Pharmacia(Piscataway, N.J., USA); prepared according to Atlas™ kit directions ofClontech) were added and the sample incubated in the dark at roomtemperature for 30 min.

The fluorescently labeled first strand cDNA was precipitated by adding 2μl 3M sodium acetate and 50 μl 100% ethanol, incubated at −20° C. for atleast 2 hrs, centrifuged at 14,000-18,000×g for 20 min, washed with 70%ethanol, air-dried for 10 min and dissolved in 100 μl of water.

Alternatively, 3-4 μg mRNA, 2.5 (˜8.9 ng of in vitro translated mRNA) μlyeast control and 3 μg oligo dTV (TTTTTTTTTTTTTTTTTT(A/C/G); Sequence IDNo.: X) were mixed in a total volume of 24.7 μl. The sample wasincubated in a thermocycler at 70° C. for 10 min. before chilling onice. To this, 8 μl of 5× first strand buffer (SuperScript II RNaseH-Reverse Transcriptase kit from Invitrogen (Carlsbad, Calif. 92008);cat no. 18064022), 0.8° C. of aa-dUTP/dNTP mix (50×; 25 mM dATP, 25 mMdGTP, 25 mM dCTP, 15 mM dTTP, 10 mM aminoallyl-dUTP), 4 μl of 0.1 M DTTand 2.5 μl (500 units) of Superscript R.T.II enzyme (Stratagene) wereadded. The sample was incubated at 42° C. for 2 hours before a mixtureof 10° C. of 1M NaOH and 10° C. of 0.5 M EDTA were added. After a 15minute incubation at 65° C., 25 μl of 1 M Tris pH 7.4 was added. Thiswas mixed with 450 μl of water in a Microcon 30 column beforecentrifugation at 11,000×g for 12 min. The column was washed twice with450 μl (centrifugation at 11,000 g, 12 min.) before eluting the sampleby inverting the Microcon column and centrifuging at 11,000×g for 20seconds. Sample was dehydrated by centrifugation under vacuum and storedat −20° C.

Each reaction pellet was dissolved in 9 μl of 0.1 M carbonate buffer(0.1M sodium carbonate and sodium bicarbonate, pH=8.5-9) and 4.5 μl ofthis placed in two microfuge tubes. 4.5 μl of each dye (in DMSO) wereadded and the mixture incubated in the dark for 1 hour. 4.5 μl of 4 Mhydroxylamine was added and again incubated in the dark for 15 minutes.

Regardless of the method used for probe generation, the probe waspurified using a Qiagen PCR cleanup kit (Qiagen, Valencia, Calif., USA),and eluted with 100 ul EB (kit provided). The sample was loaded on aMicrocon YM-30 (Millipore, Bedford, Mass., USA) spin column andconcentrated to 4-5 ul in volume. Probes for the maize microarrays weregenerated using the Fluorescent Linear Amplification Kit (cat. No.G2556A) from Agilent Technologies (Palo Alto, Calif.).

Hybridization and Wash Conditions

The following Hybridization and Washing Condition were developed:

Hybridization Conditions:

Labeled probe was heated at 95° C. for 3 min and chilled on ice. Then 25□L of the hybridization buffer which was warmed at 42 C was added to theprobe, mixing by pipetting, to give a final concentration of:

50% formamide

4×SSC

0.03% SDS

5×Denhardt's solution0.1 μg/ml single-stranded salmon sperm DNA

The probe was kept at 42 C. Prior to the hybridization, the probe washeated for 1 more min., added to the array, and then covered with aglass cover slip. Slides were placed in hybridization chambers(Telechem, Sunnyvale, Calif.) and incubated at 42° C. overnight.

Washing Conditions:

A. Slides were washed in 1×SSC+0.03% SDS solution at room temperaturefor 5 minutes,B. Slides were washed in 0.2×SSC at room temperature for 5 minutes,C. Slides were washed in 0.05×SSC at room temperature for 5 minutes.

After A, B, and C, slides were spun at 800×g for 2 min. to dry. Theywere then scanned.

Maize microarrays were hybridized according to the instructions includedFluorescent Linear Amplification Kit (cat. No. G2556A) from AgilentTechnologies (Palo Alto, Calif.).

Scanning of Slides

The chips were scanned using a ScanArray 3000 or 5000 (General Scanning,Watertown, Mass., USA). The chips were scanned at 543 and 633 nm, at 10um resolution to measure the intensity of the two fluorescent dyesincorporated into the samples hybridized to the chips.

Data Extraction and Analysis

The images generated by scanning slides consisted of two 16-bit TIFFimages representing the fluorescent emissions of the two samples at eacharrayed spot. These images were then quantified and processed forexpression analysis using the data extraction software Imagene™(Biodiscovery, Los Angeles, Calif., USA). Imagene output wassubsequently analyzed using the analysis program Genespring™ (SiliconGenetics, San Carlos, Calif., USA). In Genespring, the data was importedusing median pixel intensity measurements derived from Imagene output.Background subtraction, ratio calculation and normalization were allconducted in Genespring. Normalization was achieved by breaking the datain to 32 groups, each of which represented one of the 32 pin printingregions on the microarray. Groups consist of 360 to 550 spots. Eachgroup was independently normalized by setting the median of ratios toone and multiplying ratios by the appropriate factor.

The results of the microarray experiments are reported in the MA_DIFFTable as described above in the section entitled “Brief Description ofthe Individual Tables”.

Example 4 AFLP Experiments and Results Production of Samples

mRNA was prepared from 27 plant tissues. Based on preliminary cDNA-AFLPanalysis with a few primer combinations, 11 plant tissues and/or pooledsamples were selected. Samples were selected to give the greatestrepresentation of unique band upon electrophoresis. The final 11 samplesor pooled samples used in the cDNA-AFLP analysis were:

S1 Dark adapted seedlings S2 Roots/Etiolated Seedlings S3 Mature leaves,soil grown S4 Immature buds, inflorescence meristem S5 Flowers opened S6Siliques, all stages S7 Senescing leaves (just beginning to yellow) S8Callus Inducing medium Callus shoot induction Callus root induction S9Wounding Methyl-jasmonate-treated S10 Oxidative stress Drought stressOxygen Stress-flooding S11 Heat treated light grown seedling Coldtreated light grown seedlings

cDNA from each of the 11 samples was digested with two restrictionendonucleases, namely TaqI and MseI. TaqI and MseI adapters were thenligated to the restriction enzyme fragments. Using primers to theseadapters that were specific in sequence (i.e. without extensions), therestriction fragments were subjected to cycles of non-radioactivepre-amplification.

Selective PCR

In order to limit the number of fragments or bands on each lane of theAFLP gel, fragments were subjected to another round of selectiveradioactive polymerase chain amplification. The TaqI primers used inthis amplification were 5′-labelled with P³³. For these amplifications,the TaqI primers had two extra nucleotides at their 3′ end and the MseIprimers had three extra nucleotides at their 3′ end. This resulted in 16primer designs for the TaqI primer and 64 primer designs for the MseIprimer. Altogether, this gave rise to a total of 1024 primer designs.Fragments generated in this selective amplification protocol were runwith labeled molecular weight markers on polyacrylamide gels to separatefragments in the size range of 100-600 nucleotides.

Following gel electrophoresis, profiles were analyzed with aphosphoimager. From these images, electronic files, giving themobilities of all bands on the gels and their intensities in each of thesamples, were compiled.

All unique bands were cut out of the gels. The gel pieces were placed in96 well plates for elution and their plate designation was linked totheir electrophoretic mobilities recorded in the electronic files. Theeluted fragments were then subjected to another round of amplification,this time using reamplification primers (see below). Afteramplification, DNA fragments were sequenced.

A computer database was established linking the mobilities of all thebands observed on the cDNA-AFLP gels with the sequence of thecorrespondingly isolated fragment. The sequence allowed foridentification of the gene from which the cDNA-AFLP fragment wasderived, allowing for a linkage of band mobility with the transcript ofa specific gene. Also linked to the band mobilities were theirintensities recorded for each of the eleven samples used in constructingthe database.

This cDNA-AFLP analysis with TaqI/MseI and 1024 primer combinations wasrepeated using the enzymes NlaIII in place of TaqI, and Csp6I in placeof MseI.

Using the Database for the Transcript Profiling of Experimental Samples

Experimental Samples were subjected to cDNA-AFLP as described above,resulting in electronic files recording band mobilities and intensities.Through use of the database established above, band mobilities could belinked to specific cDNAs, and therefore genes. Furthermore, the linkagewith the intensities in the respective samples allowed for thequantification of specific cDNAs in these samples, and thus the relativeconcentration of specific transcripts in the samples, indicating thelevel to which specific genes were expressed.

Reamplification Primers

Reamplification primers 99G24CGCCAGGGTTTTCCCAGTCACGAC|ACGACTCACT|gatgagtcctgagtaa|M13 forward        +10     MseI+0 99G20AGCGGATAACAATTTCACACAGGA|CACACTGGTA|tagactgcgtaccga|M13 reverse       +10      TaqI+0

Purification of the Reamplifiction Reaction Before Sequencing

5 μl reamplification reaction0.25 μl 10×PCR buffer

0.33 μl Shrimp Alkaline Phosphatase (Amersham Life Science) 0.033 μlExonuclease I (USB)

0.297 μl SAP dilution buffer

1.59 μl MQ

7.5 μl total

30′ 37° C. 10′ 80° C. 4° C. Sample Preparation

S1: Dark adapted seedlings: Seeds of Arabidopsis thaliana(wassilewskija) were sown in pots and left at 4° C. for two to threedays to vernalize. They were transferred to a growth chamber after threedays. The intensity of light in the growth chamber was 7000-8000 LUX,temperature was 22° C., with 16 h light and 8 h dark. After 8 days, theseedlings were foil-wrapped and harvested after two days.S2: Roots/Etiolated seedlings: Seeds of Arabidopsis thaliana(wassilewskija) were germinated on solid germination media (1×MS salts,1×MS vitamins, 20 g/L sucrose, 50 mg/L MES pH 5.8) in the dark. Tissueswere harvested 14 days later.S3: Mature leaves, soil grown: Seeds of Arabidopsis thaliana(wassilewskija) were sown in pots and left at 4° C. for two to threedays to vernalize. They were transferred to a growth chamber after threedays. The intensity of light in the growth chamber was 7000-8000 LUX,temperature was 22° C., with 16 h light and 8 h dark. Leaves wereharvested 17 days later from plants that had not yet bolted.S4: Immature buds, inflorescence meristem: Seeds of Arabidopsis thaliana(wassilewskija) were sown in pots and left at 4° C. for two to threedays to vernalize. They were transferred to a growth chamber after threedays. The intensity of light in the growth chamber was 7000-8000 LUX,temperature was 22° C., with 16 h light and 8 h dark.S5: Flowers, opened: Seeds of Arabidopsis thaliana (wassilewskija) weresown in pots and left at 4° C. for two to three days to vernalize. Theywere transferred to a growth chamber after three days. The intensity oflight in the growth chamber was 7000-8000 LUX, temperature was 22° C.,with 16 h light and 8 h dark.S6: Siliques, all stages: Seeds of Arabidopsis thaliana (wassilewskija)were sown in pots and left at 4° C. for two to three days to vernalize.They were transferred to a growth chamber after three days. Theintensity of light in the growth chamber was 7000-8000 LUX, temperaturewas 22° C., with 16 h light and 8 h dark.S7: Senescing leaves (just beginning to yellow): Seeds of Arabidopsisthaliana (wassilewskija) were sown in pots and left at 4° C. for two tothree days to vernalize. They were transferred to a growth chamber afterthree days. The intensity of light in the growth chamber was 7000-8000LUX, temperature was 22° C., with 16 h light and 8 h dark. When theplant had leaves that were less than 50% yellow, the leaves that werejust beginning to yellow were harvested.

S8:

Callus Inducing Medium: Seeds of Arabidopsis thaliana (wassilewskija)were surface sterilized (1 min-75% Ethanol, 6 min-bleach 100%+Tween 20,rinse) and incubated on MS medium containing 2,4-Dichlorophenoxyaceticacid (2,4-D) 1 mg/l and Kinetin 1 mg/l in the dark for 3 weeks togenerate primary callus.

Hypocotyls and roots of the seedling were swollen after a week afterincubation in this callus induction medium and subsequently callus wasinitiated from these swollen areas.

Callus shoot induction: Primary calluses were transferred to the freshcallus induction medium for another 2 weeks growth to generate secondarycallus. Secondary callus were transferred to shoot induction mediumcontaining MS basal medium and Benzyladenine (BA) 2 mg/l andNaphthaleneacetic acid (NAA)). 1 mg/l for 2 weeks growth in the lightbefore it was harvested and frozen and sent to Keygene. Many shootmeristems were observed under the microscope.

Callus root induction: Secondary calluses were transferred to rootinduction medium containing MS basal medium, sucrose 1% andIndolebutyric acid (IBA) 0.05 mg/l in the dark. Many root primordia wereobserved under microscope after 10 days in the root induction medium.Those callus tissue were harvested and frozen and sent to Keygene.

S9:

Wounding: Seeds of Arabidopsis thaliana (wassilewskija) were sown inpots and left at 4° C. for two to three days to vernalize. They weretransferred to a growth chamber after three days. The intensity of lightin the growth chamber was 7000-8000 LUX, temperature was 22° C., with 16h light and 8 h dark. After 20 days, leaves of plants were wounded withpliers. Wounded leaves were harvested 1 hour and 4 hours after wounding.

Methyl jasmonate treatment: Seeds of Arabidopsis thaliana(wassilewskija) were sown in pots and left at 4° C. for two to threedays to vernalize. They were transferred to a growth chamber after threedays. The intensity of light in the growth chamber was 7000-8000 LUX,temperature was 22° C., with 16 h light and 8 h dark. After 13 days,plants were sprayed with 0.001% methyl jasmonate. Leaves were harvested1.5 hours and 6 hours after spraying

S10:

Oxidative stress: Seeds of Arabidopsis thaliana (wassilewskija) weresown in pots and left at 4° C. for two to three days to vernalize. Theywere transferred to a growth chamber after three days. The intensity oflight in the growth chamber was 7000-8000 LUX, temperature was 22° C.,with 16 h light and 8 h dark. After 24 days, a few leaves wereinoculated with a mixture of 2.5 mM D-glucose, 2.5 U/mL glucose oxidasein 20 mM sodium phosphate buffer pH 6.5. After an hour, 3 hours, or 5hours after inoculation, whole plant, except for the inoculated leaves,was harvested. This sample was mixed with sample from plants that weresitting in full sun (152,000 LUX) for 2 hours or four hours.

Drought stress: Seeds of Arabidopsis thaliana (wassilewskija) were sownin pots and left at 4° C. for two to three days to vernalize. They weretransferred to a growth chamber after three days. The intensity of lightin the growth chamber was 7000-8000 LUX, temperature was 22° C., with 16h light and 8 h dark. After 20 days, aerial tissues were harvested andleft to dry in 3MM Whatman paper for 1 hour or 4 hours.

Oxygen stress: Seeds of Arabidopsis thaliana (wassilewskija) were sownin pots and left at 4° C. for two to three days to vernalize. They weretransferred to a growth chamber after three days. The intensity of lightin the growth chamber was 7000-8000 LUX, temperature was 22° C., with 16h light and 8 h dark. After 21 days, the plant was flooded by immersingits pot in a beaker of tap water. After 6 days, the upper tissues wereharvested.

S11: Heat-treated light grown seedlings: Seeds of Arabidopsis thaliana(wassilewskija) were sown in pots and left at 4° C. for two to threedays to vernalize. They were transferred to a growth chamber after threedays. The intensity of light in the growth chamber was 7000-8000 LUX,temperature was 22° C., with 16 h light and 8 h dark. Over a 5 hourperiod, the temperature was raised to 42° C. at the rate ofapproximately 4° C. per hour. After 1 hour at 42° C., the aerial tissueswere collected. This sample was mixed with an equal volume of samplethat went through a heat-recovery treatment namely bringing down thetemperature to 22° C. from 42° C. over a 5 hour period at the rate of 4°C. per hour.

Cold-treated light grown seedlings: Seeds of Arabidopsis thaliana(wassilewskija) were sown in pots and left at 4° C. for two to threedays to vernalize. They were transferred to a growth chamber after threedays. The intensity of light in the growth chamber was 7000-8000 LUX,temperature was 22° C., with 16 h light and 8 h dark. After 18 days, theplant was transferred to 4° C. for an hour before the aerial tissueswere harvested. This sample was mixed with aerial tissues from anotherplant that was transferred to 4° C. for 27 hours before being harvested.

Analysis of Data:

Intensity: The intensity of the band corresponds to the value in eachlane marked S1, S2 etc.P-values: The data shows P-values of each of the samples 1-11. P-valuesare calculated using the following formula2*(1-NORMDIST(ABS(Sx-AVERAGE(of S1 to S11, not including Sx))/STDEV(ofS1 to S11 not including Sx),0,1,TRUE)) using Excel functions.

The equivalent mathematical formula of P-value is as follows:

$\mspace{20mu} {{\int{{\phi (x)}{x}}},{{integrated}\mspace{14mu} {from}\mspace{14mu} a\mspace{14mu} {to}\mspace{14mu} \infty},\mspace{20mu} {{{where}\mspace{14mu} {\phi (x)}\mspace{14mu} {is}\mspace{14mu} a\mspace{14mu} {normal}\mspace{14mu} {{distribution}:\mspace{20mu} {{where}\mspace{14mu} a}}} = \underset{\_}{{{Sx} - \mu}}}}$  σ(S 1  …  S 11, not  including  Sx);   where${\mu = {{is}\mspace{14mu} {the}\mspace{14mu} {average}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {intensities}\mspace{14mu} {of}\mspace{14mu} {all}\mspace{14mu} {samples}\mspace{14mu} {except}\mspace{14mu} {Sx}}},{= \frac{\left( {\Sigma \; S\; 1\mspace{14mu} \ldots \mspace{14mu} {Sn}} \right) - {Sx}}{n - 1}}$where  σ(S 1  …  S 11, not  including  Sx) = the  standard  deviation  of  all  sample  intensities  except  Sx.

Results:

The results are shown in the MA_diff tables.

Example 5 Transformation of Carrot Cells

Transformation of plant cells can be accomplished by a number ofmethods, as described above. Similarly, a number of plant genera can beregenerated from tissue culture following transformation. Transformationand regeneration of carrot cells as described herein is illustrative.

Single cell suspension cultures of carrot (Daucus carota) cells areestablished from hypocotyls of cultivar Early Nantes in B₅ growth medium(O. L. Gamborg et al., Plant Physiol. 45:372 (1970)) plus 2,4-D and 15mM CaCl₂ (B₅-44 medium) by methods known in the art. The suspensioncultures are subcultured by adding 10 ml of the suspension culture to 40ml of B₅-44 medium in 250 ml flasks every 7 days and are maintained in ashaker at 150 rpm at 27° C. in the dark.

The suspension culture cells are transformed with exogenous DNA asdescribed by Z. Chen et al. Plant Mol. Bio. 36:163 (1998). Briefly,4-days post-subculture cells are incubated with cell wall digestionsolution containing 0.4 M sorbitol, 2% driselase, 5 mM MES(2-[N-Morpholino]ethanesulfonic acid) pH 5.0 for 5 hours. The digestedcells are pelleted gently at 60×g for 5 min. and washed twice in W5solution containing 154 mM NaCl, 5 mM KCl, 125 mM CaCl₂ and 5 mMglucose, pH 6.0. The protoplasts are suspended in MC solution containing5 mM MES, 20 mM CaCl₂, 0.5 M mannitol, pH 5.7 and the protoplast densityis adjusted to about 4×10⁶ protoplasts per ml.

15-60 μg of plasmid DNA is mixed with 0.9 ml of protoplasts. Theresulting suspension is mixed with 40% polyethylene glycol (MW 8000, PEG8000), by gentle inversion a few times at room temperature for 5 to 25min. Protoplast culture medium known in the art is added into thePEG-DNA-protoplast mixture. Protoplasts are incubated in the culturemedium for 24 hour to 5 days and cell extracts can be used for assay oftransient expression of the introduced gene. Alternatively, transformedcells can be used to produce transgenic callus, which in turn can beused to produce transgenic plants, by methods known in the art. See, forexample, Nomura and Komamine, Plt. Phys. 79:988-991 (1985),Identification and Isolation of Single Cells that Produce SomaticEmbryos in Carrot Suspension Cultures.

Example 6 Phenotype Screens and Results A: Triparental Mating and VacuumInfiltration Transformation of Plants

Standard laboratory techniques are as described in Sambrook et al.(1989) unless otherwise stated. Single colonies of AgrobacteriumC58C1Rif, E. coli helper strain HB101 and the E. coli strain containingthe transformation construct to be mobilized into Agrobacterium wereseparately inoculated into appropriate growth media and stationarycultures produced. 100 μl of each of the three cultures were mixedgently, plated on YEB (5 g Gibco beef extract, Ig Bacto yeast extract,Ig Bacto peptone, 5 g sucrose, pH 7.4) solid growth media and incubatedovernight at 28° C. The bacteria from the triparental mating werecollected in 2 ml of lambda buffer (20 mM Tris (pH 7.5), 100 mM NaCl, 10mM MgCl₂) and serial dilutions made. An aliquot of the each dilution wasthen plated and incubated for 2 days at 28° C. on YEB platessupplemented with 100 μg/ml rifampicin and 100 μg/ml carbenicillin forcalculation of the number of acceptor cells and on YEB platessupplemented with 100 μg/ml rifampicin, 100 μg/ml carbenicillin and 100μg/ml spectinomycin for selection of transconjugant cells. Thecointegrate structure of purified transconjugants was verified viaSouthern blot hybridization.

A transconjugant culture was prepared for vacuum infiltration byinoculating 1 ml of a stationary culture arising from a single colonyinto liquid YEB media and incubating at 28° C. for approximately 20hours with shaking (220 rpm) until the OD taken at 600 nm was 0.8-1.0.The culture was then pelleted (8000 rpm, 10 min, 4° C. in a Sorvall SLA3000 rotor) and the bacteria resuspended in infiltration medium (0.5×MSsalts, 5% w/v sucrose, 10 μg/l BAP, 200 μl/l Silwet L-77, pH 5.8) to afinal OD₆₀₀ of 1.0. This prepared transconjugant culture was used within20 minutes of preparation.

Wild-type plants for vacuum infiltration were grown in 4-inch potscontaining Metromix 200 and Osmocote. Briefly, seeds of Arabidopsisthaliana (ecotype Wassilewskija) were sown in pots and left at 4° C. fortwo to four days to vernalize. They were then transferred to 22-25° C.and grown under long-day (16 hr light: 8 hr dark) conditions,sub-irrigated with water. After bolting, the primary inflorescence wasremoved and, after four to eight days, the pots containing the plantswere inverted in the vacuum chamber to submerge all of the plants in theprepared transconjugant culture. Vacuum was drawn for two minutes beforepots were removed, covered with plastic wrap and incubated in a coolroom under darkness or very low light for one to two days. The plasticwrap was then removed, the plants returned to their previous growingconditions and subsequently produced (TI) seed collected.

B: Selection of T-DNA Insertion Lines

Approximately 10,750 seeds from the initial vacuum infiltrated plantswere sown per flat of Metromix 350 soil. Flats were vernalized for fourto five days at 4° C. before being transferred to 22-25° C. and grownunder long-day (16 hr light: 8 hr dark) conditions, sub-irrigated withwater. Approximately seven to ten days after germination, the (T1)seedlings were sprayed with 0.02% Finale herbicide (AgrEvo). Afteranother five to seven days, herbicide treatment was repeated. Herbicideresistant T1 plants were allowed to self-pollinate and T2 seed werecollected from each individual. In the few cases where the T1 plantproduced few seed, the T2 seed was planted in bulk, the T2 plantsallowed to self-pollinate and T3 seed collected.

C: Phenotype Screening

Approximately 40 seed from each T2 (or T3) line were planted in a 4-inchpot containing either Sunshine mix or Metromix 350 soil. Pots werevernalized for four to five days at 4° C. before being transferred to22-25° C. and grown under long-day (16 hr light: 8 hr dark) conditions,sub-irrigated with water. A first phenotype screen was conducted byvisually inspecting the seedlings five to seven days after germinationand aberrant phenotypes noted. Plants were then sprayed with Finaleherbicide within four days (i.e. about seven to nine days aftergermination). The second visual screen was conducted on surviving T2 (orT3) plants about sixteen to seventeen days after germination and thefinal screen was conducted after the plants had bolted and formedsiliques. Here, the third and fourth green siliques were collected andaberrant phenotypes noted. The Knock-in and Knock-out Tables containdescriptions of identified phenotypes.

Alternative, seed were surface sterilized and transferred to agarsolidified medium containing Murashige and Skoog salts (1×), 1% sucrose(wt/v) pH 5.7 before autoclaving. Seed were cold treated for 48 hoursand transferred to long days [16 hours light and 8 hours dark], 25° C.Plants were screened at 5 and 10 days.

In another screen, seed were surface sterilized and transferred to agarsolidified medium containing Murashige and Skoog salts (1×), andcombinations of various nitrogen and sucrose amounts as specified below:

Medium 1: no sucrose, 20.6 mM NH₄NO₃, 18.8 mM KNO₃;

Medium 2: 0.5% sucrose, 20.6 mM NH₄NO₃, 18.8 mM KNO₃;

Medium 3: 3% sucrose, 20.6 mM NH₄NO₃, 18.8 mM KNO₃;

Medium 4: no sucrose, 20.6 μM NH₄NO₃, 18.8 μM KNO₃;

Medium 5: 0.5% sucrose, 20.6 μM NH₄NO₃, 18.8 μM KNO₃; and

Medium 6: 3% sucrose, 20.6 μM NH₄NO₃, 18.8 μM KNO₃.

The 0.5% sucrose was the control concentration for the sucrose. The lownitrogene, 20.6 μM NH₄NO₃, 18.8 μM KNO₃, is the control for thenitrogen. Seed were cold treated for 48 hours and transferred to longdays [16 hours light and 8 hours dark], 25° C. Plants were screened at2, 5, and 10 days.

D: TAIL-PCR and Fragment Sequencing

Rosette leaves were collected from each putative mutant and crushedbetween parafilm and FTA paper (Life Technologies). Two 2 mm² holepunches were isolated from each FTA sample and washed according to themanufacturer's instructions by vortexing with 200 ul of the provided FTApurification reagent. The FTA reagent was removed and the washingprocedure repeated two more times. The sample was then washed twice with200 ul of FTA TE (10 mM Tris, 0.1 mM EDTA, pH 8.0) and vortexing priorto PCR.

Primers used for TAIL-PCR are as follows:

AD2:  5′ NGTCGASWGANAWGAA 3' (128-fold degeneracy) S = G or C, W =A or T, and N = A, G,   C, or T LB1:  5′ GTTTAACTGCGGCTCAACTGTCT 3′LB2:  5′ CCCATAGACCCTTACCGCTTTAGTT 3′ LB3:  5′GAAAGAAAAAGAGGTATAACTGGTA 3′

The extent to which the left and right borders of the T-DNA insert wereintact was measured for each line by PCR. The following components weremixed for PCR: 1 2 mm² FTA sample, 38.75 μl distilled water, 5 μl 10×Platinum PCR buffer (Life Technologies), 2 μl 50 mM MgCl₂, 1 μl 10 mMdNTPs, 1 μl 10 μM primer LB1 (or RB1 for analysis of the right border),1 μl 10 μM primer LB3R (or RB3R for analysis of the right border) and1.25 U Platinum Taq (Life Technologies). Cycling conditions were: 94°C., 10 sec.; thirty cycles of 94° C., 1 sec.—54° C., 1 sec.—72° C., 1sec.; 72° C., 4 sec. The expected band size for an intact left border isbp, while an intact right border generates a bp band.

Fragments containing left or right border T-DNA sequence and adjacentgenomic DNA sequence were obtained via PCR. First product PCR reactionsuse the following reaction mixture: 1 2 mm² FTA sample, 12.44 μldistilled water, 2 μl 10× Platinum PCR buffer (Life Technologies), 0.6μl 50 mM MgCl₂, 0.4 μl 10 mM dNTPs, 0.4 μl 10 μM primer LB1 (or RB1 foranalysis of the right border), 3 μl 20 μM primer AD2 and 0.8 U PlatinumTaq (Life Technologies). Cycling conditions for these reactions were:93° C., 1 min.; 95° C., 1 min.; three cycles of 94° C., 45 sec.—62° C.,1 min.—72° C., 2.5 min.; 94° C., 45 sec.; 25° C., 3 min.; ramp to 72° C.in 3 min.; 72° C., 2.5 min.; fourteen cycles of 94° C., 20 sec.—68° C.,1 min.—72° C., 2.5 min.—94° C., 20 sec.—68° C., 1 min.—72° C., 2.5min.—94° C., 20 sec.—44° C., 1 min.—72° C., 2.5 min.; 72° C., 5 min.;end; ˜4.5 hrs. For second product PCR reactions 1 μl of a 1:50 dilutionof the first PCR product reaction was mixed with 13.44 μl distilledwater, 2 μl 10× Platinum PCR buffer (Life Technologies), 0.6 μl 50 mMMgCl₂, 0.4 μl 10 mM dNTPs, 0.4 μl 10 μM primer LB2 (or RB2 for analysisof the right border), 2 μl 20 μM primer AD2 and 0.8 U Platinum Taq (LifeTechnologies). Second product cycling conditions were: eleven cycles of94° C., 20 sec.—64° C., 1 min.—72° C., 2.5 min.—94° C., 20 sec.—64° C.,1 min.—72° C., 2.5 min.—94° C., 20 sec.—44° C., 1 min.; 72° C., 5 min.;end; ˜3 hrs. Third product PCR reactions were prepared by first diluting2 μl of the second PCR product with 98 μl of distilled water and thenadding 1 μl of the dilution to 13.44 μl distilled water, 2 μl 10×Platinum PCR buffer (Life Technologies), 0.6 μl 50 mM MgCl₂, 0.4 μl 10mM dNTPs, 0.4 μl 10 μM primer LB3 (or RB3 for analysis of the rightborder), 2 μl 20 μM primer AD2 and 0.8 U Platinum Taq (LifeTechnologies). Third product cycling conditions were: twenty cycles of94° C., 38 sec.—44° C., 1 min.—72° C., 2.5 min.; 72° C., 5 min.; end; ˜2hrs. Aliquots of the first, second and third PCR products wereelectrophoresed on 1% TAE (40 mM Tris-acetate, 1 mM EDTA) to determinetheir size.

Reactions were purified prior to sequencing by conducting a final PCRreaction. Here, 0.25 μl Platinum PCR Buffer (Life Technologies), 0.1 μl50 mM MgCl₂, 3.3 U SAP shrimp alkaline phosphatase, 0.33 U Exonucleaseand 1.781 μl distilled water were added to a 5 μl third product and thereaction cycled at 37° C., 30 min.; 80° C., 10 min.; 4° C. indefinitely.

Di-deoxy “Big Dye” sequencing was conducted on Perkin-Elmer 3700 or 377machines.

Knock-In Experiments

For the following examples, a two-component system was constructed in aplant to ectopically express the desired cDNA.

First, a plant was generated by inserting a sequence encoding atranscriptional activator downstream of a desired promoter, therebycreating a first component where the desired promoter facilitatesexpression of the activator generated a plant. The first component alsois referred to as the activator line.

Next, the second component is constructed by linking a desired cDNA to asequence that the transcriptional activator can bind to and facilitateexpression of the desired cDNA. The second component can be insertedinto the activator line by transformation. Alternatively, the secondcomponent can be inserted into a separate plant, also referred to as thetarget line. Then, the target and activator lines can be crossed togenerate progeny that have both components.

Two component lines were generated by both means.

Part I—From Crosses

Target lines containing cDNA constructs are generated using theAgrobacterium-mediated transformation. Selected target lines aregenetically crossed to activation lines (or promoter lines). Generally,the promoter lines used are as described above. Evaluation of phenotypesis done on the resulting F1 progenies.

Part II—From Type I Supertransformation

Promoter activation lines (generally Vascular/Ovule/Young Seed/Embryoline, Seed/Epidermis/Ovary/Fruit line, Roots/Shoots/Ovule line, andVasculature/Meristem are transformed with cDNA constructs using theAgrobacterium mediated transformation. Selected transformants (and theirprogenies) are evaluated for changes in phenotypes. The table for theknock-in of the Type I supertransformation comprises the followinginformation

-   -   Clone ID,    -   Pfam,    -   Gemini ID    -   Trans. Unique ID (which indicates what promoter activation line        was transformed    -   S Ratio: segregation ratio after the transformed plants are        selected for the marker.    -   Assay    -   Stage: phenotype was observed    -   Feature: Where the phenotype was observed    -   Phenotype    -   P Ratio: phenotype ratio    -   Comments

Part III—From Type II Supertransformation

Target lines generated using the procedure mentioned in Part I aretransformed with T-DNA construct containing constitutive promoter.Selected transformants (and their progenies) are evaluated for changesin phenotypes.

An additional deposit of an E. coli Library, E. coliLibAO21800, was madeat the American Type Culture Collection in Manassas, Va., USA on Feb.22, 2000 to meet the requirements of Budapest Treaty for theinternational recognition of the deposit of microorganisms. This depositwas assigned ATCC accession no. PTA-1411. Additionally, ATCC Librarydeposits; PTA-1161, PTA-1411 and PTA-2007 were made at the American TypeCulture Collection in Manassas, Va., USA on; Jan. 7, 2000, Feb. 23, 2000and Jun. 8, 2000 respectively, to meet the requirements of BudapestTreaty for the international recognition of the deposit ofmicroorganisms.

The invention being thus described, it will be apparent to one ofordinary skill in the art that various modifications of the materialsand methods for practicing the invention can be made. Such modificationsare to be considered within the scope of the invention as defined by thefollowing claims.

Each of the references from the patent and periodical literature citedherein is hereby expressly incorporated in its entirety by suchcitation.

1. An isolated nucleic acid molecule comprising: a) a nucleic acidhaving a nucleotide sequence which encodes an amino acid sequenceexhibiting at least 40% sequence identity to an amino acid sequenceencoded by (1) a nucleotide sequence described in the tables or afragment thereof; or (2) a complement of a nucleotide sequence shown inthe tables or a fragment thereof; b) a nucleic acid which is the reverseof the nucleotide sequence according to subparagraph (a), such that thereverse nucleotide sequence has a sequence order which is the reverse ofthe sequence order of the nucleotide sequence according to subparagraph(a); c) a nucleic acid capable of hybridizing to a nucleic acid having asequence selected from the group consisting of: (1) a nucleotidesequence which is shown in Tables 1 and/or 2; and a nucleotide sequencewhich is complementary to a nucleotide sequence shown in Tables 1 and/or2, under conditions that permit formation of a nucleic acid duplex at atemperature from about 40° C. and 48° C. below the melting temperatureof the nucleic acid duplex, with the proviso that said nucleotidesequence is not any of the sequences described in the Tables of any ofPatent Publication Nos. WO 200040695, CA 2300692 A1, EP 1033405 A2, CA2302828 A1 and EP 1059354 A2 and any proteins listed in the applicationthat are identified by gi number or otherwise as being from thenon-redundant GenBank CDS translations or Protein Database (PDBavailable on the internet) or PIR-International) Database (PIR;available on the internet).
 2. An isolated nucleic acid moleculecomprising a nucleic acid having a nucleotide sequence which exhibits atleast 65% sequence identity to a) a nucleotide sequence shown in thetables or a fragment thereof; or b) a complement of a nucleotidesequence described in the tables or a fragment thereof, with the provisothat said nucleotide sequence is not any of the sequences described inthe Tables of any of Patent Publication Nos. WO 200040695, CA 2300692A1, EP 1033405 A2, CA 2302828 Al and EP 1059354 A2 and any proteinslisted in the application that are identified by gi number or otherwiseas being from the non-redundant GenBank CDS translations or ProteinDatabase (PDB; available on the internet) or (PIR-International)Database (PIR) also available on the internet.
 3. The nucleic acidmolecule according to claim 1, wherein said nucleic acid comprises anopen reading frame.
 4. The isolated nucleic acid molecule of claim 1,wherein said nucleic acid is capable of functioning as a promoter, a 3′end termination sequence, an untranslated region (UTR), or as aregulatory sequence.
 5. The isolated nucleic acid molecule of claim 4,wherein (a) when said nucleic acid is a promoter it comprises a sequenceselected from the group consisting of a TATA box sequence, a CAAT boxsequence, a motif of GCAATCG or any transcription-factor bindingsequence, and any combination thereof; and (b) when said nucleic acidsequence is a regulatory sequence it is capable of promotingseed-specific expression, embryo-specific expression, ovule-specificexpression, tapetum-specific expression or root-specific expression of asequence or any combination thereof.
 6. A vector construct comprising:a) a first nucleic acid having a regulatory sequence capable of causingtranscription and/or translation; and b) a second nucleic acid havingthe sequence of the isolated nucleic acid molecule according to claim 1;wherein said first and second nucleic acids are operably linked andwherein said second nucleic acid is heterologous to any element in saidvector construct.
 7. The vector construct according to claim 6, whereinsaid first nucleic acid is native to said second nucleic acid.
 8. Thevector construct according to claim 6, wherein said first nucleic acidis heterologous to said second nucleic acid.
 9. A host cell comprisingan isolated nucleic acid molecule according to claim 1, wherein saidnucleic acid molecule is flanked by exogenous sequence.
 10. A host cellcomprising a vector construct of claim
 6. 11. An isolated polypeptidecomprising an amino acid sequence a) exhibiting at least 40%, or 75%, or85%, or 90% sequence identity of an amino acid sequence encoded by asequence shown in the tables or a fragment thereof; and b) capable ofexhibiting at least one of the biological activities of the polypeptideencoded by said nucleotide sequence shown in the tables or a fragmentthereof, with the proviso that said nucleotide sequence is not any ofthe sequences described in the Tables of any of Patent Publication Nos.WO 200040695, CA 2300692 A1, EP 1033405 A2, CA 2302828 A1 and EP 1059354A2 and any proteins listed in the application that are identified by ginumber or otherwise as being from the non-redundant GenBank CDStranslations or Protein Database (PDB; available on the internet) or(PIR-International) Database (PIR) also available on the internet. 12.An antibody capable of binding the isolated polypeptide of claim
 11. 13.A method of introducing an isolated nucleic acid into a host cellcomprising: a) providing an isolated nucleic acid molecule according toclaim 1; and b) contacting said isolated nucleic with said host cellunder conditions that permit insertion of said nucleic acid into saidhost cell.
 14. A method of transforming a host cell which comprisescontacting a host cell with a vector construct according to claim
 6. 15.A method of modulating transcription and/or translation of a nucleicacid in a host cell comprising: a) providing the host cell of claim 9;and b) culturing said host cell under conditions that permittranscription or translation.
 16. A method for detecting a nucleic acidin a sample which comprises: a) providing an isolated nucleic acidmolecule according to claim 1; b) contacting said isolated nucleic acidmolecule with a sample under conditions which permit a comparison of thesequence of said isolated nucleic acid molecule with the sequence of DNAin said sample; and c) analyzing the result of said comparison.
 17. Aplant or cell of a plant which comprises a nucleic: acid moleculeaccording to claim 1 which is exogenous or heterologous to said plant orplant cell.
 18. A plant or cell of a plant which comprises a vectorconstruct according to claim
 6. 19. A plant which has been regeneratedfrom a plant cell according to claim
 17. 20. A plant which has beenregenerated from a plant cell according to claim 18.